Droplet actuator and method

ABSTRACT

The invention provides droplet actuators and droplet actuator cassettes including reagent storage capabilities, as well as methods of making and using the droplet actuators and cassettes. The invention also provides continuous flow channel elements and techniques for using electrodes to manipulate droplets in flowing streams. The invention also discloses methods of separating compounds on a droplet actuator. Various other aspects of the invention are also disclosed.

RELATED APPLICATIONS

This application claims priority to the following U.S. PatentApplication 61/050,207, entitled “Sample collection devices with sampleprocessing and data storage capability,” filed on May 3, 2008;61/052,215, entitled “Processing Non-liquid Samples on a DropletActuator,” filed on May 11, 2008; 61/052,224, entitled “Reagent Storagefor Field-based Detection,” filed on May 11, 2008; 61/075,616, entitled“Rapid Detection of Methicillin Resistant Staphylococcus aureus (MRSA)Using Digital Microfluidics,” filed on Jun. 25, 2008; 61/085,032,entitled “Rapid Pathogen Detection on a Droplet Actuator,” filed on Jul.31, 2008; 61/088,555, entitled “Fluidic Systems for and Methods ofLoading a Droplet actuator,” filed on Aug. 13, 2008; 61/093,462,entitled Nucleic Acid Sample Preparation and Analysis on a DropletActuator,” filed on Sep. 2, 2008; and 61/157,302, entitled “DropletActuator Techniques Using Non-liquid Fluids,” filed on Mar. 4, 2009.

GOVERNMENT SUPPORT

This invention was made with government support under grant numberAI066590-02 awarded by the National Institutes of Health. The UnitedStates Government has certain rights in the invention.

The foregoing statement applies only to those aspects of the inventiondescribed and claimed in this application arising out of U.S. PatentApplication No. 61/075,616, entitled “Rapid Detection of MethicillinResistant Staphylococcus aureus (MRSA) Using Digital Microfluidics,”filed on Jun. 25, 2008; and 61/085,032, entitled “Rapid PathogenDetection on a Droplet Actuator,” filed on Jul. 31, 2008.

BACKGROUND

Droplet actuators are used to conduct a wide variety of dropletoperations. A droplet actuator typically includes two substratesseparated to form a droplet operations gap. The substrates includeelectrodes for conducting droplet operations. The gap between thesubstrates is typically filled with a filler fluid that is immisciblewith the liquid that is to be subjected to droplet operations. Dropletoperations are controlled by electrodes associated with one or both ofthe substrates. There is a need for droplet actuator devices, techniquesand systems for making and using droplet actuators. There is a need fordevices, techniques and systems for preparing samples and/or reagentsfor loading onto a droplet actuator; for loading samples and/or reagentsonto a droplet actuator; for storing samples and/or reagents on adroplet actuator and/or for use on a droplet actuator; and/or forconducting droplet operations using samples and/or reagents on a dropletactuator. There is a need for devices, techniques and systems forconducting flow through bead handling and washing. For example, there isa need for techniques for splitting droplets in a flow-through system,compartmentalizing beads in droplets in a flow-through system, andwashing droplets in a flow-through system. There is a need for dropletactuator devices, techniques and systems for making and using dropletactuators to process viscous, solid or semi-solid samples. For example,there is a need for a techniques for processing process viscous,semisolid, and/or solid samples. There is a need for droplet actuatordevices including a gel for use in gel electrophoresis, along withtechniques and systems for conducting gel electrophoresis on a dropletactuator. There is a need for a fluidics system and technique for usingthe system for loading liquids onto a droplet actuator. There is a needfor droplet actuators loaded using the fluidics system and method of theinvention and methods of using such droplet actuators to conduct dropletoperations. There is a need for droplet actuator devices, techniques andsystems for processing samples for use on a droplet actuator device.There is a need for droplet actuator devices, techniques and systems forcapturing, concentrating and/or eluting nucleic acids; and sensitivelyisolating nucleic acids using one or more droplet operations to performseparation protocols. There is a need for kits including dropletactuators of the invention along with various other components suitablefor executing the techniques of the invention, such as reagents, samplecollection devices, and/or instructions.

SUMMARY OF THE INVENTION

The invention provides a droplet actuator, and methods of making andusing the droplet actuator. The droplet actuator may include twosubstrates separated to provide a droplet operations gap. One or moreelectrodes may be associated with one or both substrates and arrangedfor conducting one or more droplet operations in the droplet operationsgap. The droplet actuator may include a reagent storage cassette withone or more reservoirs including one or more liquids. One or more fluidpaths may be provided from the one or more reservoirs into the dropletoperations gap. The fluid paths may be blocked by a film or otherbreakable, removable or puncturable material. A plunger may beassociated with the reservoir and arranged to force liquid from thereservoir into the fluid path when depressed into the reservoir. Aseries of the reservoirs and a series of the plungers may be included.Each of the reservoirs may be associated with a corresponding plungerarranged to force liquid from the reservoir into the fluid path. Theplungers may be coupled to a common plunger depressor. The film may beselected with physical and/or chemical characteristics which permit itto break upon application of pressure to liquid in the reservoir orreservoirs when the plunger or plungers are depressed. For example, filmmay be scored or include a thin or weakened region that breaks uponapplication of pressure to liquid in the reservoir or reservoirs bydepressing the plunger or plungers. The reagent storage cassette mayinclude an awl, scribe or other puncturing device arranged to puncturethe film and thereby permit liquid to flow through the fluid path. Thedevice may include an awl, point, scribe or other puncturing deviceslideably inserted within a slot in the plunger and arranged to puncturethe film and thereby permit liquid to flow through the fluid path. Thedroplet actuator may include a series of reservoirs, wherein eachreservoir in the series of the reservoirs may be associated with aconnecting fluid path extending from the reservoir and into a channel ofthe fluid path, such that upon depression of the plungers, a series ofdroplets may be forced through the connecting fluid path and into thechannel. The channel may include a liquid filler fluid which may beimmiscible with the series of droplets. The channel may be coupled to apressure or vacuum source for flowing droplets through the channel andinto the droplet operations gap. The channel may be associated with oneor more electrodes configured for transporting droplets through thechannel and into the droplet operations gap. The droplet actuator mayinclude a series of reservoirs, wherein each reservoir in the series ofreservoirs is associated with a fluid path from the reservoir into thedroplet operations gap. Liquid forced through the fluid path into thedroplet operations gap may be subject to one or more droplet operationsin the droplet operations gap. In some embodiments, the fluid path fromthe reservoir into the droplet operations gap passes through an openingin an electrode. The electrode may, for example, be a droplet operationselectrode, such as a reservoir electrode. In some embodiments, the fluidpath may be fluidly coupled with one or more filler fluid channelsarranged for flowing filler fluid around droplets in the fluid path.

The invention provides a method of conducting a droplet operationincluding providing a channel; flowing an immiscible liquid including adroplet through the channel and into proximity with a set of one or moreelectrodes; using the set of one or more electrodes with the droplet toconduct a droplet operation; and continuing to flow the droplet or oneor more daughter droplets formed during the droplet operation throughthe channel. The droplet operation may be effected without stopping flowof the immiscible liquid through the channel. The droplet operation mayinclude splitting the droplet into two or more daughter droplets. Thedroplet operation may include interrupting the flow of the dropletthrough the channel. In some embodiments, the channel splits into firstand second branches, and the droplet operation may include splitting thedroplet into two or more droplets, one or more droplets flowing into afirst of the two or more branches and a second of the two or moredroplets flowing into a second of the two or more branches. In someembodiments, the channel splits into first and second branches, and thedroplet operation causes the droplet to flow down one or the other ofthe first and second branches.

The invention also provides a method of manipulating a droplet, themethod comprising providing a channel; flowing a liquid filler fluidincluding a magnetically-responsive, bead-containing droplet through thechannel and into proximity with a magnetic field to substantiallyimmobilize the magnetically responsive bead and thereby capture thebead-containing droplet; releasing the magnetically responsive bead fromthe magnetic field, thereby permitting it to continue to flow throughthe channel. In some cases, substantially all of the liquid volume ofthe bead-containing droplet remains with the magnetically responsivebead when it may be substantially immobilized by the magnetic field. Inother cases, at least a portion of the liquid volume of thebead-containing droplet breaks away from the magnetically responsivebead when it may be substantially immobilized by the magnetic field andcontinues to flow with the liquid filler fluid through the channel. Themethod may also include flowing a second droplet in the flowing fillerfluid into contact with the captured bead-containing droplet, whereinthe second droplet merges with the bead-containing droplet. In someembodiments the flowing filler fluid causes a bead-free droplet to breakaway from the bead-containing droplet and continue to flow with theliquid filler fluid through the channel. The second droplet may, forexample, include a wash buffer. The method may also include repeatingthe flowing of a second droplet in the flowing filler fluid into contactwith the captured bead-containing droplet using a series of two or moreof such second droplets to reduce the concentration and/or quantity of asubstance present in the liquid volume of the bead-containing droplet.The method may also include repeating the flowing of a second droplet inthe flowing filler fluid into contact with the captured bead-containingdroplet until the liquid volume of the bead-containing droplet may besubstantially replaced. The second droplet may include a sample droplethaving a target for which the bead may have affinity. The method mayalso include repeating the flowing of a second droplet in the flowingfiller fluid into contact with the captured bead-containing dropletusing a series of two or more of such second droplets to concentrate atarget substance on the bead of the bead-containing droplet. The targetsubstance may, for example, include organic molecules, inorganicmolecules, peptides, proteins, macromolecules, subcellular components ofa biological cell, cells, group of cells, single celled organisms,multicellular organisms. The method may also include flowing a thirddroplet in the flowing filler fluid into contact with the capturedbead-containing droplet, wherein the third droplet merges with thebead-containing droplet; the flowing filler fluid causes a bead-freedroplet to break away from the bead-containing droplet and continue toflow with the liquid filler fluid through the channel. The third dropletmay, for example, include a wash buffer. The method may also includerepeating the flowing of a third droplet in the flowing filler fluidinto contact with the captured bead-containing droplet using a series oftwo or more of such third droplets sufficient to reduce theconcentration and/or quantity of a substance present in the liquidvolume of the bead-containing droplet. The method may also includerepeating the flowing of a third droplet in the flowing filler fluidinto contact with the captured bead-containing droplet until the liquidvolume of the bead-containing droplet may be substantially replaced. Themethod may also include releasing the magnetically responsive bead fromthe magnetic field, e.g., to permit a reconstituted magneticallyresponsive bead containing droplet to flow in the filler fluid throughthe channel. In some embodiments the magnetic field may be in proximitywith a set of one or more electrodes and the method may include usingthe set of one or more electrodes with the bead-containing droplet toconduct a droplet operation. The method may include continuing to flowthe droplet or one or more daughter droplets formed during the dropletoperation through the channel. The method may include flowing thereconstituted magnetically responsive bead containing droplet into adroplet actuator reservoir and/or into a droplet operations gap of adroplet actuator, where the magnetically responsive bead containingdroplet may be subjected to one or more droplet operations. The one ormore droplet operations may include steps in an assay protocol toanalyze a target substance on the magnetically responsive bead. Thedroplet operation may be effected without stopping flow of theimmiscible liquid through the channel. The droplet operation may includesplitting the droplet into two or more daughter droplets: one or more ofsuch daughter droplets including the magnetically responsive bead; andone or more of such daughter droplets substantially lacking inmagnetically responsive beads. The droplet operation may includeinterrupting the flow of the droplet through the channel. In some cases,the channel splits into first and second branches; and the dropletoperation includes splitting the droplet into two or more droplets,including one or more daughter droplets flowing into a first of the twoor more branches and including the magnetically responsive bead; and oneor more daughter droplets flowing into a second of the two or morebranches and substantially lacking in magnetically responsive beads.

The invention also provides a method of encapsulating a magneticallyresponsive bead in a droplet, the method may include providing achannel; flowing an immiscible liquid including a magneticallyresponsive bead through the channel and into proximity with a magneticfield; capturing the magnetically responsive bead in the magnetic field;flowing an immiscible liquid including a droplet into contact with themagnetically responsive bead to encapsulate the magnetically responsivebead in the droplet, thereby yielding a bead-containing droplet. Themagnetically responsive bead may have affinity for an aqueous medium,the droplet may include an aqueous medium, and the filler fluid mayinclude a non-aqueous liquid. The method may also include releasing themagnetically responsive bead from the magnetic field, thereby permittingthe bead-containing droplet to continue to flow with the filler fluidthrough the channel.

The invention provides a method of sampling a non-liquid sample on adroplet actuator, the method may include providing a droplet actuatorincluding a droplet operations and electrodes configured to conduct oneor more droplet operations on the droplet operations surface; supplyinga non-liquid sample in proximity to or in contact with the dropletoperations surface; effecting one or more droplet operations to contacta droplet on the droplet operations surface into contact with thenon-liquid sample to dissolve into the droplet one or more components ofthe non-liquid sample; effecting one or more droplet operations toconduct the droplet away from the non-liquid sample. The non-liquidsample may include a solid sample, a semi-solid sample and/or a viscoussample. The droplet may include one or more beads having affinity forone or more of the components of the non-liquid sample. The droplet mayinclude an enzyme having affinity for a component of the non-liquidsample. The droplet may have pH selected to dissolve the non-liquidsample. The sample may include cells and the droplet may include a lysisbuffer solution selected to lyse the cells. The one or more dropletoperations may include an electrode-mediated droplet operation. The oneor more droplet operations may include an electrowetting-mediateddroplet operation. The one or more droplet operations may include adielectrophoresis-mediated droplet operation. The non-liquid samplesufficiently viscous, semi-solid or solid to permit a droplet to contactthe sample and be transported away from the sample without beingsubstantially combined with the sample. The non-liquid sample may beselected from the group consisting of sputum, coagulated blood, animaltissue samples, plant tissue samples, soil samples, and rock samples.The non-liquid sample may include a matrix used to collect the sample.The droplet may include an aqueous droplet. The droplet may include anon-aqueous droplet. The method may include using the droplet to conductan assay analyzing a component of the sample. In some cases, the assayanalyzes a protein or peptide present in the sample. In some cases, theassay may include amplifying a nucleic acid present in the sample. Themethod may include removing the droplet from the droplet actuator.

The invention provides a method of providing a polymerized material on adroplet operations surface. The method may include providing a dropletactuator including a substrate including electrodes arranged forconducting droplet operations on a droplet operations surface of thesubstrate. The method may include providing on the droplet operationssubstrate a polymerizable droplet on the droplet operations substrateand a catalyst droplet including a catalyst selected to acceleratepolymerization of the polymerizable droplet. The method may also includeconducting droplet operations mediated by the electrodes to combine thepolymerizable droplet with the catalyst droplet to yield a polymerizingdroplet. Further, the method may include permitting the polymerizingdroplet to polymerize, thereby yielding a polymerized material on thedroplet operations surface. The droplet actuator may include a secondsubstrate separated from the droplet operations surface to provide adroplet operations gap in which the droplet operations may be conducted.In some embodiments the droplet operations may be conducted in a liquidfiller fluid which may be immiscible with the polymerizable droplet andthe catalyst droplet. In some embodiments the polymerized material mayinclude a gel selected for conducting gel electrophoresis. Thepolymerized material may, for example, be a polyacrylamide gel or anagarose gel. The method may include activating a series of two or moreelectrodes underlying the polymerizable droplet to elongate the dropletprior to combining the polymerizable droplet with the catalyst droplet.The method may include activating a series of two or more electrodesunderlying the polymerizing droplet to elongate the droplet prior topermitting the polymerizing droplet to polymerize.

The invention also provides a method of causing separation of one ormore substances. The method may include providing a sample dropletincluding substances for separation on the droplet operations surface orin a reservoir associated with a fluid path arranged to flow liquid fromthe reservoir into contact with the polymerized material. The method mayinclude contacting the sample droplet with the polymerized material. Themethod may include applying current to the polymerized material to causeseparation of one or more of the substances provided in the sampledroplet. In some cases, the droplet actuator may include a secondsubstrate separated from the droplet operations surface to provide adroplet operations gap, the second substrate including an openingproviding a fluid path from an exterior locus into the dropletoperations gap; and providing a sample droplet may include supplying asample droplet through the opening in the second substrate into contactwith the polymerized material. The method may include marking one ormore target substances for detection. For example, in some cases, theone or more substances for separation may include one or more nucleicacids, and the marking may include staining the one or more nucleicacids. In some embodiments the marking may include providing a markerdroplet on the droplet operations surface, and using one or more dropletoperations to transport the marker droplet into contact with thepolymerized material.

The invention also provides a droplet actuator including a substrateincluding electrodes arranged for conducting droplet operations on adroplet operations surface of the substrate; a polymerized material forconducting gel electrophoresis; negative and positive electrodes incontact with the polymerized material. The droplet actuator may includea second substrate separated from the droplet operations surface toprovide a droplet operations gap in which the droplet operations may beconducted. The droplet actuator may include a liquid filler fluid incontact with the droplet operations surface. The polymerized materialmay, for example, include a gel selected from the group consisting ofpolyacrylamide gels and agarose gels. The droplet actuator may include asample droplet including substances for separation on the dropletoperations surface or in a reservoir associated with a fluid patharranged to flow liquid from the reservoir into contact with thepolymerized material. The substances for separation may, for example,include proteins, peptides and/or nucleic acids. The droplet actuatormay include a second substrate separated from the droplet operationssurface to provide a droplet operations gap. In some cases, the secondsubstrate including an opening providing a fluid path from an exteriorlocus into the droplet operations gap. The droplet actuator may includeincluding a marker droplet including reagents for marking one or moretarget substances in the polymerized material for detection.

The invention provides a droplet actuator loading circuit including aprimary fluid circuit arranged to flow fluid through a fluid pathincluding a droplet operations gap of a droplet actuator and a anexternal fluid circuit. The droplet actuator loading circuit may includea reagent fluid path branching from the primary fluid circuit andfluidly connecting the primary fluid path to one or more reservoirsincluding reagents and/or filler fluid. The droplet actuator loadingcircuit may include a mechanism for switching the reagent fluid pathfrom one reservoir to another reservoir. The mechanism for switching thereagent fluid path between reagent reservoirs may include a roboticdevice for moving a terminus of the reagent fluid path from onereservoir to another reservoir. The droplet actuator loading circuit mayinclude one or more valves configured in the primary fluid circuitand/or the reagent fluid path to permit switching between circulatingliquid in the primary fluid circuit, and flowing liquid from the one ormore reservoirs including reagents and/or filler fluid into the primaryfluid circuit. The droplet actuator loading circuit may include areagent fluid path branching from the primary fluid circuit and fluidlyconnecting the primary fluid path to one or more reservoirs includingreagents, and a filler fluid path branching from the primary fluidcircuit and fluidly connecting the primary fluid path to one or morereservoirs including a liquid filler fluid. The droplet actuator loadingcircuit may include one or more valves configured in the primary fluidcircuit and/or the reagent fluid path to permit switching betweencirculating liquid in the primary fluid circuit, and flowing liquidreagent from the one or more reservoirs including reagents into theprimary fluid circuit, flowing filler fluid from the one or morereservoirs including liquid filler fluid into the primary fluid circuit.The droplet actuator loading circuit, wherein the reagent fluid pathand/or the filler fluid path branches from the primary fluid circuit ata locus which may be in the external fluid circuit. The droplet actuatorloading circuit including a pump disposed to pump liquid through theprimary fluid circuit. The pump may, for example, include a reversiblepump. The pump may include a peristaltic pump. The pump may be disposedto pump liquid through the primary fluid circuit, wherein the pump maybe disposed in the primary fluid circuit at a locus which lies between alocus in the primary fluid circuit at which the reagent fluid pathbranches from the primary fluid circuit, and a locus in the primaryfluid circuit at which the filler fluid path branches from the primaryfluid circuit. The droplet actuator loading circuit may also include anoverflow fluid path fluidly coupled into the droplet operations gap. Thedroplet actuator loading circuit may also include a reservoir and a pumpdisposed to pump liquid from the droplet operations gap through theoverflow fluid path and into a reservoir. The reservoir and pumptogether may comprise a syringe pump.

The invention provides a method of loading a droplet actuator. Themethod may include providing a droplet actuator loading circuitincluding a primary fluid circuit arranged to flow fluid through a fluidpath including a droplet operations gap of a droplet actuator and a anexternal fluid circuit. The method may include filling the loadingcircuit, including the droplet operations gap, with a liquid fillerfluid and thereby purging the loading circuit of air. The method mayinclude flowing reagent liquid into the external fluid circuit to formdroplets in the liquid filler fluid contained therein. The method mayinclude flowing contents of the external fluid circuit into the dropletoperations gap of the droplet actuator. Filling the loading circuit,including the droplet operations gap, with a liquid filler fluid mayinclude flowing filler fluid into the primary fluid circuit via a fillerfluid branch in the primary fluid circuit. The filler fluid branch inthe primary fluid circuit may be situated in the external fluid circuit.Flowing reagent liquid into the external fluid circuit may includeflowing reagent into the primary fluid circuit via a reagent branch inthe primary fluid circuit. The reagent branch in the primary fluidcircuit may be situated in the external fluid circuit. Different kindsof reagent droplets may be loaded into the external fluid circuit.Reagent types may be selected by switching the reagent branch from onereservoir to another reservoir. The switching may be effected by arobotic device configured to move a terminus of the reagent fluid pathfrom one reservoir to another reservoir. Valves configured in theprimary fluid circuit and/or the reagent fluid path to switch betweencirculating liquid in the primary fluid circuit, and flowing liquid fromthe one or more reservoirs including reagents and/or filler fluid intothe primary fluid circuit. The method further may include flowing liquidfrom the droplet operations gap through an overflow fluid path fluidlycoupled into the droplet operations gap. Flowing liquid from the dropletoperations gap through an overflow fluid path may include pumping theliquid through the overflow path into a reservoir. The reservoir andpump together may include a syringe pump.

The invention provides a method of preparing a sample droplet. Themethod may include providing a droplet actuator substrate including adroplet operations surface and electrodes configured to conduct dropletoperations on the droplet operations surface. The method may includeproviding a sample droplet including cells including a target substanceon the droplet operations surface. The method may include providing alysis droplet including a lysis buffer on the droplet operationssurface. The method may include using one or more droplet operationsmediated by the electrodes to combine the lysis droplet with the sampledroplet to yield a lysed droplet including lysed cells. The method mayinclude providing in the lysed droplet beads having affinity for thetarget substance. The sample droplet may include beads. Beads may beadded to the sample droplet buffer prior to providing the sample dropleton the droplet operations surface. The sample droplet may be merged witha bead droplet including the beads on the droplet operations surface.The lysis droplet may be provided with the beads. Beads may be added tothe lysis buffer prior to providing the lysis droplet on the dropletoperations surface. The lysis droplet may be merged with a bead dropletincluding the beads on the droplet operations surface. The method mayinclude washing the beads to yield a washed bead droplet substantiallylacking in unbound material from the sample. The method may includeproviding an elution droplet on the droplet operations surface, andusing one or more droplet operations to combine the elution droplet withthe washed bead droplet to yield an eluted droplet in which the targetsubstance may be eluted from the beads. The method may include heatingthe combined elution droplet and washed bead droplet to accelerateelution of target substance from the beads. The method may includetrapping the beads and using one or more droplet operations to transportaway from the beads a substantially bead-free droplet on the dropletoperations surface. The target substance may include a target protein ortarget peptide. The target substance may include a target nucleic acid.The method may include supplying the substantially bead-free droplet orthe washed bead droplet with reagents for conducting nucleic acidamplification to yield an amplification-ready droplet. One or moredroplet operations may be used to combine the substantially bead-freedroplet or the washed bead droplet with an amplification reagent dropletincluding reagents for conducting nucleic acid amplification. The methodmay include thermal cycling the amplification-ready droplet to amplifythe target nucleic acid. The cells may include eukaryotic cells orprokaryotic cells. The cells may include bacterial cells. In someembodiments, the bacterial cells may include cells from Staphylococcusspecies, Streptococcus species, Enterococcus species, Pseudomonasspecies, Clostridium species, and/or Acinetobacter species. In someembodiments, the bacterial cells may include cells from Staphylococcusaureus, Pseudomonas aeruginosa, Clostridium difficile, Acinetobacterbaumannii, Bacillus anthracis, Franciscella tularensis, Mycoplasmapneumoniae, and Eschericia coli.

The invention provides a droplet actuator device including a dropletactuator including a electronic storage and/or transmission element. Theelectronic storage and/or transmission element may be affixed to orincorporated in a droplet actuator. The electronic storage and/ortransmission element may be affixed to or incorporated in a dropletactuator cartridge including a droplet actuator. The electronic storageand/or transmission element may include a computer readable data storageelement. The computer readable data storage element may includesemiconductor memory, magnetic storage, optical storage, volatilememory, non-volatile memory, a radio-frequency identification tag,read-only memory, random access memory, electrically erasableprogrammable read-only memory, flash memory, and/or a magnetic stripe.The magnetic stripe may be provided on a magnetic stripe card, and thedroplet actuator may be mounted on the magnetic stripe card. The dropletactuator mounted on the magnetic stripe card may include electricalcontacts arranged to couple with electrical contacts on a dropletactuator instrument when the magnetic stripe card may be inserted in amagnetic card slot of a magnetic card reading instrument. The dropletactuator may be electrically connected to wires on the card. The wireson the card may terminate in contacts arranged to be electricallycoupled to electrical contacts on an instrument so that the dropletactuator may be controlled by the instrument. The card may have a shapeand size of a standard credit card. The electronic storage and/ortransmission element may include a unique identifier for the dropletactuator. The droplet actuator device may be configured with a connectdevice for connecting the droplet actuator device to a computer as aperipheral device. The connect device may, for example, include auniversal serial bus connector. The droplet actuator device may alsoinclude a positioning device, such as a global positioning device.

The invention also includes a networked system including the dropletactuator device distributed in a target geographical region withcommunications capabilities for transmitting data to one or more dataaggregation centers. The droplet actuators may be installed on fixedbases. The fixed bases may be selected from the group consisting of:buildings, farms, water supply sources, buoys, and weather balloons. Thedroplet actuators may be installed on fixed bases. The mobile bases maybe selected from the group consisting of: mobile robotic devices,airplanes, unmanned drones, vehicles in vehicle fleets. The mobile basesmay be selected from the group consisting of: police cars, school buses,ambulances, military vehicles, oceangoing vessels, postal vehicles, andvehicles in commercial vehicle fleets.

The invention provides a system including the droplet actuator deviceand a global position sensor. The invention provides a system includingone or more kiosks including a dispenser for dispensing a dropletactuator device. The invention provides a system including one or morekiosks including a receptacle for receiving a droplet actuator device.The kiosk may also include an input device for inputting informationassociated with a droplet actuator device.

DEFINITIONS

As used herein, the following terms have the meanings indicated.

“Activate” with reference to one or more electrodes means effecting achange in the electrical state of the one or more electrodes which, inthe presence of a droplet, results in a droplet operation.

“Bead,” with respect to beads on a droplet actuator, means any bead orparticle capable of interacting with a droplet on or in proximity with adroplet actuator. Beads may be any of a wide variety of shapes, such asspherical, generally spherical, egg shaped, disc shaped, cubical andother three dimensional shapes. The bead may, for example, be capable ofbeing transported in a droplet on a droplet actuator or otherwiseconfigured with respect to a droplet actuator in a manner which permitsa droplet on the droplet actuator to be brought into contact with thebead, on the droplet actuator and/or off the droplet actuator. Beads maybe manufactured using a wide variety of materials, including forexample, resins, and polymers. The beads may be any suitable size,including for example, microbeads, microparticles, nanobeads andnanoparticles. In some cases, beads are magnetically responsive; inother cases beads are not significantly magnetically responsive. Formagnetically responsive beads, the magnetically responsive material mayconstitute substantially all of a bead or one component only of a bead.The remainder of the bead may include, among other things, polymericmaterial, coatings, and moieties which permit attachment of an assayreagent. Examples of suitable magnetically responsive beads aredescribed in U.S. Patent Publication No. 2005-0260686, entitled,“Multiplex flow assays preferably with magnetic particles as solidphase,” published on Nov. 24, 2005, the entire disclosure of which isincorporated herein by reference for its teaching concerningmagnetically responsive materials and beads. The fluids may include oneor more magnetically responsive and/or non-magnetically responsivebeads. Examples of droplet actuator techniques for immobilizingmagnetically responsive beads and/or non-magnetically responsive beadsand/or conducting droplet operations protocols using beads are describedin U.S. patent application Ser. No. 11/639,566, entitled “Droplet-BasedParticle Sorting,” filed on Dec. 15, 2006; U.S. Patent Application No.61/039,183, entitled “Multiplexing Bead Detection in a Single Droplet,”filed on Mar. 25, 2008; U.S. Patent Application No. 61/047,789, entitled“Droplet Actuator Devices and Droplet Operations Using Beads,” filed onApr. 25, 2008; U.S. Patent Application No. 61/086,183, entitled “DropletActuator Devices and Methods for Manipulating Beads,” filed on Aug. 5,2008; International Patent Application No. PCT/US2008/053545, entitled“Droplet Actuator Devices and Methods Employing Magnetic Beads,” filedon Feb. 11, 2008; International Patent Application No.PCT/US2008/058018, entitled “Bead-based Multiplexed Analytical Methodsand Instrumentation,” filed on Mar. 24, 2008; International PatentApplication No. PCT/US2008/058047, “Bead Sorting on a Droplet Actuator,”filed on Mar. 23, 2008; and International Patent Application No.PCT/US2006/047486, entitled “Droplet-based Biochemistry,” filed on Dec.11, 2006; the entire disclosures of which are incorporated herein byreference.

“Droplet” means a volume of liquid on a droplet actuator that is atleast partially bounded by filler fluid. For example, a droplet may becompletely surrounded by filler fluid or may be bounded by filler fluidand one or more surfaces of the droplet actuator. Droplets may, forexample, be aqueous or non-aqueous or may be mixtures or emulsionsincluding aqueous and non-aqueous components. Droplets may take a widevariety of shapes; nonlimiting examples include generally disc shaped,slug shaped, truncated sphere, ellipsoid, spherical, partiallycompressed sphere, hemispherical, ovoid, cylindrical, and various shapesformed during droplet operations, such as merging or splitting or formedas a result of contact of such shapes with one or more surfaces of adroplet actuator. For examples of droplet fluids that may be subjectedto droplet operations using the approach of the invention, seeInternational Patent Application No. PCT/US 06/47486, entitled,“Droplet-Based Biochemistry,” filed on Dec. 11, 2006. In variousembodiments, a droplet may include a biological sample, such as wholeblood, lymphatic liquid, serum, plasma, sweat, tear, saliva, sputum,cerebrospinal liquid, amniotic liquid, seminal liquid, vaginalexcretion, serous liquid, synovial liquid, pericardial liquid,peritoneal liquid, pleural liquid, transudates, exudates, cystic liquid,bile, urine, gastric liquid, intestinal liquid, fecal samples, liquidscontaining single or multiple cells, liquids containing organelles,fluidized tissues, fluidized organisms, liquids containing multi-celledorganisms, biological swabs and biological washes. Moreover, a dropletmay include a reagent, such as water, deionized water, saline solutions,acidic solutions, basic solutions, detergent solutions and/or buffers.Other examples of droplet contents include reagents, such as a reagentfor a biochemical protocol, such as a nucleic acid amplificationprotocol, an affinity-based assay protocol, an enzymatic assay protocol,a sequencing protocol, and/or a protocol for analyses of biologicalfluids.

“Droplet Actuator” means a device for manipulating droplets. Forexamples of droplet actuators, see U.S. Pat. No. 6,911,132, entitled“Apparatus for Manipulating Droplets by Electrowetting-BasedTechniques,” issued on Jun. 28, 2005 to Pamula et al.; U.S. patentapplication Ser. No. 11/343,284, entitled “Apparatuses and Methods forManipulating Droplets on a Printed Circuit Board,” filed on filed onJan. 30, 2006; U.S. Pat. No. 6,773,566, entitled “ElectrostaticActuators for Microfluidics and Methods for Using Same,” issued on Aug.10, 2004 and U.S. Pat. No. 6,565,727, entitled “Actuators forMicrofluidics Without Moving Parts,” issued on Jan. 24, 2000, both toShenderov et al.; Pollack et al., International Patent Application No.PCT/US2006/047486, entitled “Droplet-Based Biochemistry,” filed on Dec.11, 2006; and Roux et al., U.S. Patent Pub. No. 20050179746, entitled“Device for Controlling the Displacement of a Drop Between two orSeveral Solid Substrates,” published on Aug. 18, 2005; the disclosuresof which are incorporated herein by reference. Certain droplet actuatorswill include a substrate, droplet operations electrodes associated withthe substrate, one or more dielectric and/or hydrophobic layers atop thesubstrate and/or electrodes forming a droplet operations surface, andoptionally, a top substrate separated from the droplet operationssurface by a gap. One or more reference electrodes may be provided onthe top and/or bottom substrates and/or in the gap. In variousembodiments, the manipulation of droplets by a droplet actuator may beelectrode mediated, e.g., electrowetting mediated or dielectrophoresismediated or Coulombic force mediated. Examples of other methods ofcontrolling liquid flow that may be used in the droplet actuators of theinvention include devices that induce hydrodynamic fluidic pressure,such as those that operate on the basis of mechanical principles (e.g.external syringe pumps, pneumatic membrane pumps, vibrating membranepumps, vacuum devices, centrifugal forces, piezoelectric/ultrasonicpumps and acoustic forces); electrical or magnetic principles (e.g.electroosmotic flow, electrokinetic pumps, ferrofluidic plugs,electrohydrodynamic pumps, attraction or repulsion using magnetic forcesand magnetohydrodynamic pumps); thermodynamic principles (e.g. gasbubble generation/phase-change-induced volume expansion); other kinds ofsurface-wetting principles (e.g. electrowetting, and optoelectrowetting,as well as chemically, thermally, structurally and radioactively inducedsurface-tension gradients); gravity; surface tension (e.g., capillaryaction); electrostatic forces (e.g., electroosmotic flow); centrifugalflow (substrate disposed on a compact disc and rotated); magnetic forces(e.g., oscillating ions causes flow); magnetohydrodynamic forces; andvacuum or pressure differential. In certain embodiments, combinations oftwo or more of the foregoing techniques may be employed in dropletactuators of the invention.

“Droplet operation” means any manipulation of a droplet on a dropletactuator. A droplet operation may, for example, include: loading adroplet into the droplet actuator; dispensing one or more droplets froma source droplet; splitting, separating or dividing a droplet into twoor more droplets; transporting a droplet from one location to another inany direction; merging or combining two or more droplets into a singledroplet; diluting a droplet; mixing a droplet; agitating a droplet;deforming a droplet; retaining a droplet in position; incubating adroplet; heating a droplet; vaporizing a droplet; cooling a droplet;disposing of a droplet; transporting a droplet out of a dropletactuator; other droplet operations described herein; and/or anycombination of the foregoing. The terms “merge,” “merging,” “combine,”“combining” and the like are used to describe the creation of onedroplet from two or more droplets. It should be understood that whensuch a term is used in reference to two or more droplets, anycombination of droplet operations that are sufficient to result in thecombination of the two or more droplets into one droplet may be used.For example, “merging droplet A with droplet B,” can be achieved bytransporting droplet A into contact with a stationary droplet B,transporting droplet B into contact with a stationary droplet A, ortransporting droplets A and B into contact with each other. The terms“splitting,” “separating” and “dividing” are not intended to imply anyparticular outcome with respect to volume of the resulting droplets(i.e., the volume of the resulting droplets can be the same ordifferent) or number of resulting droplets (the number of resultingdroplets may be 2, 3, 4, 5 or more). The term “mixing” refers to dropletoperations which result in more homogenous distribution of one or morecomponents within a droplet. Examples of “loading” droplet operationsinclude microdialysis loading, pressure assisted loading, roboticloading, passive loading, and pipette loading. Droplet operations may beelectrode-mediated. In some cases, droplet operations are furtherfacilitated by the use of hydrophilic and/or hydrophobic regions onsurfaces and/or by physical obstacles.

“Filler fluid” means a liquid associated with a droplet operationssubstrate of a droplet actuator, which liquid is sufficiently immisciblewith a droplet phase to render the droplet phase subject toelectrode-mediated droplet operations. The filler fluid may, forexample, be a low-viscosity oil, such as silicone oil. Other examples offiller fluids are provided in International Patent Application No.PCT/US2006/047486, entitled, “Droplet-Based Biochemistry,” filed on Dec.11, 2006; International Patent Application No. PCT/US2008/072604,entitled “Use of additives for enhancing droplet actuation,” filed onAug. 8, 2008; and U.S. Patent Publication No. 20080283414, entitled“Electrowetting Devices,” filed on May 17, 2007; the entire disclosuresof which are incorporated herein by reference. The filler fluid may fillthe entire gap of the droplet actuator or may coat one or more surfacesof the droplet actuator. Filler fluid may be conductive ornon-conductive.

“Immobilize” with respect to magnetically responsive beads, means thatthe beads are substantially restrained in position in a droplet or infiller fluid on a droplet actuator. For example, in one embodiment,immobilized beads are sufficiently restrained in position to permitexecution of a splitting operation on a droplet, yielding one dropletwith substantially all of the beads and one droplet substantiallylacking in the beads.

“Magnetically responsive” means responsive to a magnetic field.“Magnetically responsive beads” include or are composed of magneticallyresponsive materials. Examples of magnetically responsive materialsinclude paramagnetic materials, ferromagnetic materials, ferrimagneticmaterials, and metamagnetic materials. Examples of suitable paramagneticmaterials include iron, nickel, and cobalt, as well as metal oxides,such as Fe3O4, BaFe12O19, CoO, NiO, Mn2O3, Cr2O3, and CoMnP.

“Washing” with respect to washing a magnetically responsive bead meansreducing the amount and/or concentration of one or more substances incontact with the magnetically responsive bead or exposed to themagnetically responsive bead from a droplet in contact with themagnetically responsive bead. The reduction in the amount and/orconcentration of the substance may be partial, substantially complete,or even complete. The substance may be any of a wide variety ofsubstances; examples include target substances for further analysis, andunwanted substances, such as components of a sample, contaminants,and/or excess reagent. In some embodiments, a washing operation beginswith a starting droplet in contact with a magnetically responsive bead,where the droplet includes an initial amount and initial concentrationof a substance. The washing operation may proceed using a variety ofdroplet operations. The washing operation may yield a droplet includingthe magnetically responsive bead, where the droplet has a total amountand/or concentration of the substance which is less than the initialamount and/or concentration of the substance. Examples of suitablewashing techniques are described in Pamula et al., U.S. Pat. No.7,439,014, entitled “Droplet-Based Surface Modification and Washing,”granted on Oct. 21, 2008, the entire disclosure of which is incorporatedherein by reference.

The terms “top,” “bottom,” “over,” “under,” and “on” are used throughoutthe description with reference to the relative positions of componentsof the droplet actuator, such as relative positions of top and bottomsubstrates of the droplet actuator. It will be appreciated that thedroplet actuator is functional regardless of its orientation in space.

When a liquid in any form (e.g., a droplet or a continuous body, whethermoving or stationary) is described as being “on”, “at”, or “over” anelectrode, array, matrix or surface, such liquid could be either indirect contact with the electrode/array/matrix/surface, or could be incontact with one or more layers or films that are interposed between theliquid and the electrode/array/matrix/surface.

When a droplet is described as being “on” or “loaded on” a dropletactuator, it should be understood that the droplet is arranged on thedroplet actuator in a manner which facilitates using the dropletactuator to conduct one or more droplet operations on the droplet, thedroplet is arranged on the droplet actuator in a manner whichfacilitates sensing of a property of or a signal from the droplet,and/or the droplet has been subjected to a droplet operation on thedroplet actuator.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate a reagent storage cassette of the invention.FIG. 1A shows the reagent storage cassette and an open position. FIG. 1Bshows the reagent storage cassette in a closed position.

FIGS. 2A and 2B illustrate a reagent storage cassette of the inventionincluding a protective film configured to protect reagents fromcontamination and/or prevent leaking of reagents. FIG. 2A shows thereagent storage cassette and an open position. FIG. 2B shows analternative embodiment of the reagent storage cassette in a closedposition where the film includes a pull tab.

FIGS. 3A, 3B, 3C, 3D, and 3E show cross-sectional views of segments oftop and bottom members of a reagent storage cassette of the inventionthat makes use of plungers to force droplets from storage reservoirs.FIG. 3A illustrates a segment of the bottom member with the protectivefilm in place. FIG. 3B illustrates the segment of the bottom member withplungers fully inserted to force droplets from storage reservoirs. FIGS.3C and 3D illustrate a corresponding segment of a top member of areagent storage cassette juxtaposed with the cross sectional view of asegment of the bottom member of the reagent storage cassette. In FIG.3C, the droplets are present in the reservoirs. In FIG. 3D, plungers arefully inserted to force droplets from storage reservoirs. FIG. 3Eillustrates an end-wise cross-sectional view of the reagent storagecassette showing a section of the top member juxtaposed with acorresponding section of the bottom member.

FIG. 4 illustrates another embodiment of the invention in which adroplet actuator cartridge is provided with a droplet actuator portionan integral reagent cassette portion.

FIGS. 5A and 5B illustrate a side cross-sectional view and a top view,respectively, of a droplet actuator configured to supply droplets intoreservoirs in a droplet operations gap.

FIGS. 6A, 6B, 6C, 6D, 6E, and 6F illustrate another aspect of theinvention in which a plunger is used to force liquid into the dropletoperations gap of a droplet actuator. FIG. 6A shows liquid in areservoir outside the droplet operations gap, prior to being forced intothe droplet operations gap. FIG. 6B shows a top view of a reservoirelectrode through which liquid is supplied into the droplet operationsgap of the droplet actuator. FIG. 6C illustrates puncturing thedielectric layer so that liquid may flow into the droplet operationsgap. FIG. 6D illustrates the plunger fully inserted and liquid havingbeen forced into the droplet operations gap. FIG. 6E shows analternative embodiment in which a single electrode is provided on a topsubstrate of the droplet actuator. FIG. 6F illustrates an alternativeembodiment in which a reservoir electrode and droplet operationselectrodes are provide on the top substrate and a ground or referenceelectrode is provided on the bottom substrate.

FIG. 7 illustrates an alternative embodiment of the reagent cassette ofthe invention including a first channel into which droplets are loaded,and a second channel for flowing liquid filler fluid around the dropletsin the first channel.

FIG. 8 illustrates a flow-through system that makes use of dropletoperations for splitting droplets.

FIG. 9 illustrates a flow-through system configured for adding beads todroplets.

FIG. 10 illustrates a flow-through system that makes use of dropletoperations to wash beads in droplets.

FIGS. 11A, 11B, and 11C illustrate a section of a droplet actuator and amethod of processing a viscous, solid or semi-solid sample on a dropletactuator.

FIGS. 12A, 12B, and 12C illustrate a section of a droplet actuator and aprocess of separating and analyzing a sample using gel electrophoresis.

FIGS. 13A, 13B, 13C, 13D, 13E, 13F, 13G, 13H, and 13I are schematicdiagrams of fluidics system for loading liquid receptacle, such as achannel or droplet operations gap of a droplet actuator, with liquid.FIG. 13A illustrates the system generally, while FIGS. 13B-13I eachillustrate a specific step in a loading process.

FIGS. 14A, 14B, 14C, 14D, 14E, and 14F are schematic diagrams of anotherfluidics system for loading liquid receptacle, such as a channel ordroplet operations gap of a droplet actuator, with liquid. FIG. 14Aillustrates the system generally, while FIGS. 14B-14F each illustrate aspecific step in a loading process.

FIG. 15A shows a plot of real-time PCR data for detection of MRSA usingdigital microfluidics. FIG. 15B shows a plot of real-time PCR data fordetection of Bacillus anthracis using digital microfluidics.

FIG. 16 shows a plot resulting from amplification of MRSA genomic DNAcaptured, concentrated and eluted on a droplet actuator.

FIG. 17 illustrates a droplet actuator device of the invention,including a droplet actuator with an electronic storage and/ortransmission element.

FIG. 18 illustrates another droplet actuator device of the invention,including a droplet actuator with an electronic storage and/ortransmission element, where the electronic storage and/or transmissionelement includes a magnetic stripe card.

FIG. 19 is a functional diagram of a sample collection and analysissystem of the invention.

FIG. 20 is a functional diagram of another sample collection andanalysis system of the invention.

DESCRIPTION

The invention provides droplet actuator devices, techniques and systemsfor making and using droplet actuators. The invention provides devices,techniques and systems for preparing samples and/or reagents for loadingonto a droplet actuator; for loading samples and/or reagents onto adroplet actuator; for storing samples and/or reagents on a dropletactuator and/or for use on a droplet actuator; and/or for conductingdroplet operations using samples and/or reagents on a droplet actuator.The invention also provides devices, techniques and systems forconducting flow through bead handling and washing. For example, theinvention provides techniques for splitting droplets in a flow-throughsystem, compartmentalizing beads in droplets in a flow-through system,and washing droplets in a flow-through system. The invention providesdroplet actuator devices, techniques and systems for making and usingdroplet actuators to process viscous, solid or semi-solid samples. Forexample, the invention provides techniques for processing viscous,semisolid, and/or solid samples. The invention provides droplet actuatordevices including a gel for use in gel electrophoresis, along withtechniques and systems for conducting gel electrophoresis on a dropletactuator. The invention provides a fluidics system and technique forusing the system for loading liquids onto a droplet actuator. Theinvention also provides droplet actuators loaded using the fluidicssystem and method of the invention and methods of using such dropletactuators to conduct droplet operations. The invention provides dropletactuator devices, techniques and systems for processing samples for useon a droplet actuator device. In some cases, the processing includespre-processing steps conducted prior to introduction of the samples ontoa droplet actuator. The invention provides droplet actuator devices,techniques and systems for capturing, concentrating and/or elutingnucleic acids; and sensitively isolating nucleic acids using one or moredroplet operations to perform separation protocols. The invention alsoprovides kits including droplet actuators of the invention along withvarious other components suitable for executing the techniques of theinvention, such as reagents, sample collection devices, and/orinstructions.

Liquid Storage and Loading

The invention provides devices, techniques and systems for preparingsamples and/or reagents for loading onto a droplet actuator; for loadingsamples and/or reagents onto a droplet actuator; for storing samplesand/or reagents on a droplet actuator and/or for use on a dropletactuator; and/or for conducting droplet operations using samples and/orreagents on a droplet actuator. The reagents may be stored on thedroplet actuator itself, and/or in reagent storage containers that areprovided with a droplet actuator cartridge. In some cases, the dropletactuator cartridge may be provided in a kit along with reagents storedand storage containers.

Reagents selected for storage in accordance with the invention may bereagents which are useful in conducting an assay. For example, thereagents may be useful in an assay for assessing the presence or absenceof, and/or quantify the amount of, a chemical or a biochemicalsubstance. Examples of suitable assay types include immunoassays,nucleic acid amplification assays, nucleic acid sequencing assays,enzymatic assays, and other forms of assays. Assays may be conductedwith various purposes; examples include medical diagnostics, veterinarydiagnostics, weapons or explosives detection, chemical weaponsdetection, biological weapons detection, environmental testing, watertesting, air testing, soil testing, food quality testing, forensics,species identification etc.

Samples may be collected and tested at a point of sample collection. Forexample, the point of sample collection may be in a medical carefacility, at a subject's bedside, in a laboratory, or in the field. Asample may be collected, loaded onto the droplet actuator cartridge; thecartridge may be inserted into an instrument, an assay may be run, andresults may be provided, all at the point of sample collection. In otherembodiments, one or more of these steps may be accomplished remotelyfrom the point of sample collection, e.g., in a central laboratory. Asample may be collected in the field, and transported to a laboratory,where it is loaded into a cartridge which is mounted on an instrument;the assay may be run, and results provided. A sample may be collected inthe field and loaded into a cartridge in the field, e.g., loaded into asample reservoir in a droplet actuator cartridge; the cartridge may bereturned to a laboratory, where it is mounted on an instrument, an assayis run, and results are provided. Various other combinations are alsopossible within the scope the invention. The instrument may includeelectronic and detection components, as well as a means for mounting thecartridge on the instrument, or otherwise coupling the cartridge to theinstrument, in a manner in which aligns electronic and detectioncomponents with corresponding components or regions of the dropletactuator. The cartridge may include a droplet actuator, electricalcomponents which correspond to the electrical components of theinstrument, and one or more detection regions, which are aligned withdetection components on the instrument. In various embodiments, thereagent storage and loading techniques described in this specificationmay also be used for loading sample, e.g., sample may be collected andloaded into a reservoir, where it is stored. Sample may be loaded fromthe reservoir into the droplet operations gap of the droplet actuatorcartridge in preparation for conducting an assay using the dropletactuator cartridge.

The reagent storage and reconstitution techniques of the invention maybe useful in a variety of fluidics devices, such as droplet actuatordevices. In some cases, the devices of the invention are packaged withor include reagents. The reagents may be provided in a format that issuitable for use in the field. In certain embodiments, the devices aresuitable for use without requiring refrigeration and/or specializeddispensing equipment. Reagents may be rapidly reconstituted and used inexpress testing. Assay results, currently available by in-laboratorytesting, may be made available substantially in ‘real-time.’ Decisionsmade using assay results can be made more quickly.

FIG. 1 illustrates a reagent storage cassette 100 of the invention.Cassette 100 includes a bottom member 105 and a top member 110. Bottommember 105 and a top member 110 may be coupled by a flexible hingeportion or member 115. FIG. 1A shows bottom member 105 and a top member110 in an open position. FIG. 1B shows bottom member 105 and a topmember 110 in a closed position. In one embodiment, the storage cassetteis provided with bottom member 105 and a top member 110 in an openposition, and the cassette is manipulated by a user into the closedposition shown in FIG. 1B. The flexible member 115 may have differentelectrical and physical properties in the regions overlying bottommember 105, overlying top member 110, and at the hinge and thus cancomprise of multiple materials or a same material with differentproperties or a same material with substantially similar properties. Theflexible member 115 may be an insulating material that serves dualpurpose of insulating the electrodes on substrate 105 that are used foreffecting droplet operations and also serve as a tether to the topplate. The flexible member 115 may have different properties in theportions overlying members 105 and 110 so that it is insulating onbottom member 105 but substantially conductive on top member 110 or viceversa. In other embodiments, the flexible member 115 may be rigid overmembers 105 and 110 but flexible only at the hinge connecting both themembers. In another embodiment, the flexible member may also comprise agasket or standoff material that forms a gap between the members 105 and110 so that droplets can reside between members 105 and 110. Theflexible member may also be hydrophobized so that it is ready fordroplet operations.

FIG. 2 illustrates a reagent storage cassette 100 of the invention withprotective film 205. Protective film 205 may be included to provide aseal, enclosing reagent components to protect them from the environmentand/or separating reagent components from one another. One embodiment,shown in FIG. 2A illustrates a protective film 205 covering facingsurfaces of the bottom member 105 and a top member 110 of cassette 100.In operation, protective film 205 is removed to expose reagentcomponents, and then bottom member 105 and a top member 110 of cassette100 are sealed together. FIG. 2B illustrates a protective film insertedbetween facing surfaces of the bottom member 105 and a top member 110 ofcassette 100. In operation, protective film 205 is removed to exposereagent components in bottom member 105 to reagent components in topmember 110 of cassette 100. The protective film can also serve as aremovable insulating or hydrophobic material. After droplet operationsare performed, the members 110 and 105 may be separated and a new film205 may be attached so that the surfaces of the droplet actuator areclean and can be reused without any concern for cross contamination.

FIG. 3 illustrates cross-sectional views 300 of a segment of bottommember 105. Bottom member 105 includes reservoir 305 formed therein,which serve as reservoirs for droplets 315 of liquid reagents orsamples. Openings 310 provide a liquid path for forcing droplets 315onto a surface of bottom member 105. During storage, protective film205, illustrated in FIG. 3A, may be maintained in place to seal droplets315 in reservoirs 310. Droplets 315 may be pre-metered; however, in somecases, exact premetering is not required, since the droplets will besubject to dispensing operations on the droplet actuator in whichdispensed subdroplets will have precise volumes suitable for conductingassays.

Reservoir 305 is associated with plungers 325 and plunger depressor 330.Plunger depressor 330 is configured to force plungers 325 into openings310, thereby forcing droplets 315 out of openings 310. Plunger depressor330 may be manually operated, such that an operator may, by applyingpressure to plunger depressor 330, force plungers 325 into openings 310,thereby forcing droplets 315 out of openings 310. Plunger depressor 330may be automatically operated, for example, so that when an operatorinserts the droplet actuator cartridge into an instrument, the activeinsertion also forces plunger depressor 330 to move plungers through 25into openings 310. While reservoir/plunger assemblies are illustrated inFIGS. 3A/3B, it will be appreciated that in some cases only a singlesuch reservoir/plunger assembly is required. In other cases, more thantwo reservoir/plunger assemblies may be provided.

Prior to forcing plungers 325 into openings 310, protective film 205 maybe removed. Alternatively, protective film 205 may be scored orotherwise weakened in regions atop openings 310 so that by applyingpressure to plunger depressor 330, droplets 315 may be forced out ofopenings 310 and through protective film 205. In yet another embodiment,an awl, scribe, needle, or other puncturing component may be used topuncture or weaken protective film 205. For example, top member 110 maybe equipped with an awl, scribe or other component configured topuncture or weaken protective film 205 when bottom member 105 and topmember 110 are fitted together.

One or more of droplets 315 may be a fully constituted reagent. One ormore of droplets 315 may, when forced out of opening 310, contact andcombine with one or more reagents on surface of bottom member 105 and/ortop member 110 of cassette 100 to yield a fully constituted reagent. Inanother embodiment, droplet 315 constitutes a sample. For example, asample may be loaded in reservoir 305 at a point of sample collection,and may later be loaded in accordance with the reagent loadingtechniques described herein. In another embodiment, droplet 315constitutes a standard solution with known amount of material that caneither be used as a calibrant or can be diluted using droplet operationsto setup a standard curve using multiple concentrations derived fromperforming dilutions.

In some embodiments, protective film 205 also serves as an adhesiveand/or dielectric layer. For example, a droplet actuator substrate 105may include electrodes (not shown) associated with substrate 105.Protective film 205 may be a dielectric layer atop the electrodes,arranged such that the electrodes may be used to conduct dropletoperations atop protective film 205. Protective film 205 may or may notbe bound to substrate 105 using an adhesive layer. A hydrophilic coating(not shown) may, in some cases, be provided atop protective film 205.

Substrate 105 may be any rigid substrate, such as a silicon, PCB,plastic, or other polymeric substrate. Electrodes may be any materialwhich is suitably conductive to permit electrodes to mediate dropletoperations atop protective film 205. Examples include copper, chrome,aluminum, gold, silver, indium tin oxide, and other conductivematerials. The adhesive layer, when present, may be any adhesive whichis suitable for binding protective film 205 to the underlying layers ofsubstrate 105. In alternative embodiments, the adhesive layer may beabsent. Protective film 205 may be any dielectric material, andhydrophobic coating may be any hydrophobic coating that binds to theunderlying layers in a manner which is sufficient to permit one or moredroplet operations to be conducted atop droplet actuator substrate 105.The protective film 205 may be coated with a hydrophobic layer. Examplesof suitable hydrophobic coatings include fluoropolymers andperfluoroploymers, such as polytetrafluoroethylenes; perfluoroalkoxypolymer resins; fluorinated ethylene-propylenes;polyethylenetetrafluoroethylenes; polyvinylfluorides;polyethylenechlorotrifluoroethylenes; polyvinylidene fluorides;polychlorotrifluoroethylenes; and perfluoropolyethers. In oneembodiment, the hydrophobic coating includes an amorphous Teflonfluoropolymer or a TEFLON® fluoropolymer. In another embodiment, thehydrophobic coating includes a CYTOP™ perfluoropolymer.

In one embodiment, an adhesive layer binds protective film 205 toelectrodes and substrate 105. In one example, protective film 205 is apolyimide film. In yet another example, the adhesive layer includes anacrylic adhesive. In still another example, an adhesive-backed polyimidefilm provides adhesive layer and protective film 205. For example,adhesive-backed polyimide film may be a PYRALUX® LF flexible composite(DuPont). PYRALUX® LF7013, for example is an approximately 13 μMviscous, solid or semi-solid DuPont KAPTON® polyimide film and 25 μMviscous, solid or semi-solid acrylic adhesive. Other examples ofsuitable adhesive-backed films include PYRALUX® LF LF0110, LF0120,LF0130, LF0150, LF0210, LF0220, LF0230, LF0250, LF0310, LF7001, LF7082,LF1510, and LF7034.

In some embodiments, the adhesive is selected to be releasable, so thatthe adhesive-backed film may be removed following use and replaced witha fresh adhesive-backed film. In some embodiments, the adhesive mayserve as the protective film and the backing may serve as a hydrophobiccoating. In other embodiments, the dielectric may be formed as apermanent part of the substrate, and a protective film having ahydrophilic backing may be applied to the permanent dielectric. In yetanother embodiment, multiple films may be used and replaced together orseparately. For example, a hydrophobic film may be used atop aprotective film, and both films may be applied atop a droplet actuatorsubstrate including electrodes. Each of the hydrophobic film andprotective film may be replaced together or separately, as needed.

In one embodiment, the protective film includes a dielectric film, andthe droplet actuator substrate includes the substrate, electrodes and adielectric atop the substrate. The protective film is placed atop thedielectric, and an adhesive may optionally be included between thedielectric and the protective film.

In another embodiment, the protective film includes a dielectric film,and the droplet actuator substrate includes the substrate, electrodesand a dielectric atop the substrate. The protective film may be placedatop the dielectric, and an adhesive may optionally be included betweenthe dielectric and the protective film. Alternatively, the dropletactuator substrate may include the substrate and electrodes with nodielectric atop the substrate. The protective film may be placed atopthe substrate and electrodes, and an adhesive may optionally be includedbetween the substrate and electrodes and the film.

Top member 110 may be equipped with an awl, scribe or other componentconfigured to puncture or weaken protective film 205 when bottom member105 and top member 110 are fitted together. Plungers 325 may be equippedwith an awl, scribe or other component configured to puncture or weakenprotective film 205 when plungers 325 are inserted in reservoirs 305.Once liquid 315 is forced atop substrate 105, electrodes associated withtop member 110 and/or bottom member 105 may be used to effect dropletoperations using droplets 315. In certain embodiments, the awl, scribeor other component configured to puncture or weaken protective film hasa hydrophobic surface.

In yet another embodiment, the protective film may double as ahydrophobic layer. For example, the wells may be located in the topsubstrate and separated from the gap by the protective film, doubling asa hydrophobic layer. The protective film may be punctured during loadingof droplets into the gap, e.g., by an awl, scribe or other componentconfigured to puncture or weaken protective film, permitting droplets toflow through the punctured region and into the gap where they aresubject to droplet operations. In certain embodiments, the awl, scribeor other component configured to puncture or weaken protective film hasa hydrophobic surface.

In an alternative embodiment, the top substrate and bottom substrate areprovided bound together, and separated to provide a droplet operationsgap. In this embodiment, the droplets 315 would be forced by theplungers 325 into the gap, where they would be subject to dropletoperations using electrodes associated with the top member 110 and/orthe bottom member 105.

FIGS. 3C and 3D illustrates a length-wise cross sectional view 301 of asegment of top member 110 juxtaposed with a cross sectional view 300 ofa segment of bottom member 105 described with reference to FIG. 3B. Asshown in FIG. 3C, top member 110 includes dried reagent 375 affixedthereto. When droplet 315 is forced out of its opening and into contactwith dried reagent 375, droplet 315 combines with dried reagent 375 toyield a fully constituted reagent, as illustrated in FIG. 3D.

FIG. 3E illustrates an end-wise cross-sectional view of reagent storagecassette 302 showing top member 110 juxtaposed with bottom member 105.Top member 110 includes channel 306, which may be any type of liquidpath. Bottom member 105 includes reservoirs 305 formed therein. Film 205seals channel 306 and reservoir 305. Reservoir 305 includes a liquid,such as a sample or a reagent or a calibrant. Cassette 302 may includemultiple reservoirs. Channel 306 may include one or more dried,concentrated or viscous, solid or semi-solidened reagents 375. Eachdried, concentrated or viscous, solid or semi-solidened reagent 375 maybe aligned with a corresponding reservoir 305, such that when liquid 315is caused to flow into channel 306, each dried reagent 375 is combinedwith a droplet of liquid 315 to yield a constituted reagent. Reservoir305 is associated with plungers 325 and plunger depressor 330. Plungerdepressor 330 is configured to permit a user to force plungers 325 intoreservoir 305, thereby forcing droplets 315 out of openings 310 and intochannel 306. Reservoir 305 is thus bounded and substantially sealed bysubstrate 105 along opening 310, film 205 and plunger 325. In someembodiments, compressible material 380 may be provided between plungercompressor 330 and bottom member 105 to retain plunger 325 in placeduring storage and shipment.

In operation, film 205 may be removed. Plunger 325 may be forced intoreservoir 305, thereby forcing liquid 315 into channel 306. A series ofsuch liquids 315 may be forced into channel 306, thereby forming aseries of droplets separated by a filler fluid. In cases where thereagent storage cassette is provided as an integral part of a dropletactuator cartridge, droplets 315 may be transported and from the reagentstorage cassette into another region of the cartridge. For example,reagent droplets 315 may be transported from the reagent storagecassette into a droplet operations gap of a droplet actuator. Similarly,droplets 315 may be transported from the reagent storage cassette into areservoir of a droplet actuator, from where they may be transportedthrough a liquid path into a droplet operations gap of a dropletactuator. In the droplet operations gap, droplets 315 and/orsub-droplets dispensed therefrom may be subjected to droplet operations.For example, the droplet operations may be part of a droplet operationsprotocol which is designed to use droplets 315 to perform an assay.

In some embodiments, a pressure source may provide pressure for forcingdroplets from the reagent storage cassette into a droplet actuator, orinto another region of a droplet actuator cassette. As illustrated inFIG. 3D, a pressure source may forced droplets 315 through channel 306and into the droplet actuator. Channel 306 may be in any configuration,for example, it may be linear or curvilinear. Channel 306 may beprovided generally in a common plane with a droplet operations gap, suchthat droplets 315 may flow along a common plane through channel 306 andinto the droplet operations gap. Alternatively, channel 306 may beprovided in a different plane than the plane of the droplet operationsgap. For example, channel 306 may be located in a plane which isseparate, but parallel to the playing of the droplet operations gap. Aliquid passage may connect channel 306 to the droplet operations gap,such that the pressure source may cause the droplets 315 to flow throughchannel 306, through the connecting liquid passage, and into the dropletoperations gap. In yet another embodiment, the channel 306 need not beparallel to the droplet operations gap, e.g., the channel may be in aposition relative to the droplet operations gap which establishes anangle which is between 0 and 180°.

In one embodiment, pressure may be applied to the contents of channel306, thereby forcing droplets and filler fluid into a droplet operationsgap of the droplet actuator. Alternatively, a vacuum source may be usedto pull the contents of channel 306 into a droplet operations gap of adroplet actuator. Further, channel 306 may itself be associated withelectrodes capable of effecting forces suitable for causing thetransport of droplets 315 along the path of channel 306. Such electrodesmay, for example, form a path having one or more electrode members whichare adjacent to electrode members in a droplet operations gap of adroplet actuator. In this manner, the electrodes may be used totransport one or more droplets through channel 306, and from channel 306into a droplet operations gap of a droplet actuator.

FIG. 4 illustrates another embodiment of the invention in which adroplet actuator cartridge 400 is provided with a droplet actuatorportion 401 and an integral reagent cassette portion 402. In theembodiment illustrated, droplet actuator portion 401 includes topsubstrate 410 separated from bottom substrate 415 by droplet operationsgap 420. Bottom substrate 415 includes electrodes 418 arranged forconducting one of more droplet operations in droplet operations gap 420.It will also be appreciated that one or more droplet operations and/orreference electrodes may be associated with top substrate 410 and/orbottom substrate 415. Reagent cassette portion 402 includes bottomsubstrate 410, which is the same as top substrate 410 of dropletactuator portion 401. Reagent cassette portion 402 also includes topsubstrate 405, which includes reservoirs 435 formed therein. Asillustrated, plungers 325 are inserted into reservoirs 435. Channel 306is formed in top substrate 405 and/or bottom substrate 410. Channel 306is connected to droplet actuator gap 420 by liquid path 440. Channel 306may also be coupled to a pressure source 445 configured for providingpressure into channel 306. Pressure source 445 may be coupled to channel306 by a liquid path 448 established, for example, by capillary tube 450and associated fitting 455. Similarly, an output flow path 460 may becoupled to droplet operations gap 420; the coupling may, for example, beestablished by a capillary tube 465 and associated fitting 470. In thismanner, a liquid path is established from pressure source 445 throughliquid path 448, through channel 306, through connecting liquid path440, through droplet operations gap 420, and through exit liquid path460. In some cases, rather than a pressure source 445, a vacuum source480 may be coupled via liquid path 460 to droplet operations gap 420. Inoperation, droplets 315 may be stored in reservoirs 435. A film (notshown) may be provided over openings to reservoirs 435 to retaindroplets 315 therein. As noted above, the film may be scored in order tofacilitate breaking of the film upon application of pressure thereto byinsertion of plungers 325. Alternatively, a puncturing device may beemployed, e.g., as illustrated below with respect to FIG. 6. In anycase, plungers 325 may be forced into reservoirs 435, thereby forcingdroplets 315 into channel 306. Pressure from pressure source 445 and/orvacuum from vacuum source 480 may be used to cause droplets 315 to flowthrough channel 306, through liquid path 440, and into dropletoperations gap 420 where such droplets may be subject to dropletoperations mediated by electrodes 418. In an alternative embodiment,droplet operations and/or reference electrodes may be associated withsurfaces adjacent to channel 306 and/or connecting liquid path 440, anddroplets 315 may be transported into droplet operations gap using one ormore droplet operations facilitated by such electrodes. The figure isillustrative, and many other embodiments are possible. For example, FIG.4 can be considered as a top view (top plate not shown and 410 serves asonly a gasket with no electrodes) where the plungers are inserted withinthe gap of the droplet actuator and electrodes 418 move to underneaththe channel 420 while 415 serves as a gasket.

FIGS. 5A and 5B illustrate a side cross-sectional view and a top view,respectively, of a droplet actuator 500 according to the invention.Droplet actuator 500 is like droplet actuator 400, except that ratherthan forcing droplets into a channel, which is used to supply dropletsinto a droplet operations gap, droplet actuator 500 supplies dropletsdirectly into reservoirs in a droplet operations gap. One or moresub-droplets may be dispensed from the reservoirs. Droplet actuator 500includes top substrate 505 and bottom substrate 510, separated by gasket515 to form gap 520. Top substrate 505 includes droplet operationselectrodes 523, though it will be appreciated that as describedelsewhere herein, in an alternative embodiment, droplet operationselectrodes 523 may be supplied on bottom substrate 510 rather than topsubstrate 505. Bottom substrate 510 also includes reservoirs 525 intowhich plungers 530 are inserted. Gasket 515 also forms reservoirs 535 indroplet operations gap 520. Each reservoir 535 includes an electrode 523associated with top substrate 505 and aligned with reservoir 535.Adjacent to each electrode 523 is a path of droplet operationselectrodes 524. The paths of droplet operations electrodes 524 arearranged in a network of paths. It will be appreciated that the networkof paths illustrated in FIG. 5B is illustrative only, and that a widevariety of similar such networks is possible within the scope of theinvention. FIG. 5A shows droplets 536, including one droplet in eachreservoir 535. As illustrated, the droplets have been forced in theplace using plungers 530, i.e., by forcing plungers 530 into reservoirs525. In operation, droplet actuator 500 may include a protective film asdescribed herein, which may be removed and/or punctured prior to forcingdroplets 536 into place within reservoirs 535. Ideally, the top surfaceof each plunger is hydrophobic or is coated with a hydrophobic materialin order to facilitate droplet operations conducted using electrodes 523and 524. Further, the surface of reservoir 525 and/or reservoir 535 mayalso be hydrophobic or coated with a hydrophobic material.

FIGS. 6A, 6B, 6C, 6D, 6E, and 6F illustrate another aspect of theinvention in which a plunger is used to force liquid into the dropletoperations gap of a droplet actuator. As illustrated in FIG. 6A, dropletactuator 600 includes a top substrate 605 and a bottom substrate 610separated by gasket 615 the form drop operations gap 620. Reservoir 625is formed on bottom substrate 610. As illustrated, reservoir 625includes a liquid 630, which may, for example, include reagent and/orsample. Bottom substrate 610 further includes electrodes 635 arrangedfor conducting droplet operations in the droplet operations gap. Bottomsubstrate 610 further includes an electrode 636, which includes anopening 637 therein. FIG. 6A shows liquid 630 in reservoir 625 outsidethe droplet operations gap 620, prior to being forced by plunger 650into the droplet operations gap 620.

FIG. 6B shows a top view of electrode 636. Opening 637 is shown as beingcentrally located, but it will be appreciated that the opening may beprovided in any region of electrode 636. Further, in an alternativeembodiment, no opening is provided in electrode 636, and instead, anopening providing a liquid path into the droplet operations gap isprovided adjacent to electrode 636. It will also be appreciated thatwhile the opening is shown as being generally circular, any shape issuitable. Moreover, while a single opening is shown, multiple openingsmay be provided. Opening 637 provides a liquid path 645 from reservoir625 into droplet operations gap 620.

Bottom substrate 610 further includes a dielectric layer 640 atopelectrodes 635 and 636. Dielectric layer 640 blocks the liquid path.Various examples of a dielectric layer are as described above withrespect to aspects in which protective film 205 is a dielectric layer. Ahydrophobic layer may also be provided atop dielectric layer 640.Droplet actuator 600 also includes a plunger 650, which extends intoreservoir 625, and seals liquid 630 therein. Plunger 650 includes an awl655 arranged for puncturing dielectric layer 640 to open liquid path645, thereby permitting liquid 630 to flow through liquid path 645 andinto droplet operations gap 620. As illustrated, awl 655 is insertedthrough an opening in plunger 650 and aligned to puncture dielectriclayer 640 through opening 637.

Puncturing of dielectric layer 640 is illustrated in FIG. 6C. Onpuncturing dielectric layer 640, plunger 650 may be forced intoreservoir 625, thereby forcing liquid 630 through liquid path 645 intodroplet operations gap 620.

FIG. 6D illustrates plunger 650 in a fully inserted position, and showsliquid 630 situated in droplet operations gap 620 atop electrode 636.From this position, droplets may be dispensed from liquid 630 usingelectrodes 635 and 636. Awl 655 is shown in a retracted position inwhich the tip of awl 655 is removed from the punctured region ofdielectric 640 in order to permit liquid to flow with reducedobstruction through liquid path 645.

FIG. 6E shows droplet actuator 601, which is like droplet actuator 600,except that in droplet actuator 601, a single electrode 660 is providedon top substrate 605. Electrode 660 may serve as a reference electrode.In one embodiment, top substrate 605 is made from a transparentmaterial, such as glass or plastic, while electrode 660 is also madefrom a transparent electrode material, such as indium tin oxide.

FIG. 6F shows droplet actuator 602, which is like droplet actuator 601in FIG. 6E, except that in droplet actuator 602, a reservoir electrode636 and droplet operations electrodes 635 are provided on top substrate605, while ground or reference electrode 660 is provided on bottomsubstrate 610. Fluid 630 flows into droplet operations gap 620 throughliquid path 645, which includes an opening 637 in ground electrode 660.

In other embodiments, the plunger 650 is not required and only theawl/needle 655 is utilized. As shown in FIG. 6D, plunger 650 serves as afixed element and an integral part of bottom substrate 610 and in somecases they both may be the same element. The needle 655 in this case maybe actuated during the action of loading the cartridge. The needlepuncturing the dielectric 640 and part of the electrode 636 may behydrophilic so that upon puncturing the liquid automatically is drawnonto electrode 636. In another embodiment, the needle and the reservoirarrangement could be on the top plate 605.

FIG. 7 illustrates an alternative embodiment of the reagent cassette ofthe invention. In addition to the components already described, thisembodiment includes a channel 705 for flowing an immiscible liquidfiller fluid around droplets 315 and/or droplets 605. Further, topmember includes a top plunger member 710, which may be used to agitatedroplet 315 in the presence of dried reagent 481 to promote mixing. Topplunger member 710 may be associated with a sonicator arranged tovibrate top plunger member 710 and thereby promote mixing of driedreagent 481 in droplet 315.

Steps A-F illustrate the following: Step A shows top member 305 andbottom member 405 with protective film 205 in place, protecting droplets315 and dried reagent 481. Step B shows top member 305 and bottom member405 with protective film 205 removed, and top member 305 and bottommember 405 fitted together. Step C shows plunger 325 compressed to forcedroplet 315 into channel 505. Step D shows droplet 315 mixed with driedreagent 418. Step E shows filler fluid flowed through channels 705 intospace in channel 505 surrounding droplets 315. Step F shows compressionof plunger member 710 to compress droplet 315, e.g., to cause mixing ofdroplet 315.

Various embodiments may include a filler fluid reservoir in associationwith channel 705 and/or channel 505 for flowing oil into channel 505. Inother embodiments, droplets 315 may include beads and/or dried reagentsmay include beads which dissolve into droplets 315. Beads may, forexample, have affinity for target analytes or compounds that interferewith assay chemistry. Some embodiments may include vents from channel705 and/or channel 505 for venting bubbles prior to loading dropletsonto a droplet actuator or other microfluidic device. Protective filmsmay be made from any material which is suitably non-reactive withreagents contacting the films. Examples include aluminum and variouspolymeric films. Dried reagents for use in the cassette may be preparedusing methods known to one of skill in the art, such as commercialoff-the-shelf (COTS) equipment and well-established procedures.

Flow Through Bead Handling and Washing Techniques

The invention also provides devices, techniques and systems forconducting flow through bead handling and washing. For example, theinvention provides techniques for splitting droplets in a flow-throughsystem, compartmentalizing beads in droplets in a flow-through system,and washing droplets in a flow-through system.

FIG. 8 illustrates a flow-through system 800 that makes use of dropletoperations for splitting droplets. Flow-through system 800 includeschannel 805 which intersects with channel 810. A set of electrodes 812are associated with channel 810 at a position which is approximatelyopposite to an entry point of channel 805 into channel 810. The internalwalls of channels 805 and 810 are hydrophobic. Channels 805 and 810 arefilled with a liquid filler fluid which is substantially immiscible withparent droplets 815. Parent droplets 815 flow through channel 805 in thedirection of arrow A. When electrodes 812 are activated, the internalwall of channel 810 in the region of electrodes 812 behaves inhydrophilic manner. When a parent droplet 815 impacts theelectrode-associated region, the droplet spreads to conform to the shapeof the activated electrodes. When an intermediate electrode isdeactivated, the droplet splits into two sub-droplets 816. In theembodiment illustrated, the two sub-droplets 816 flow into channel 810in opposite directions, as illustrated by arrows B and C. In operation,by controlling the flow of filler fluid through channels 805 and 810,droplets 815 may be sequentially contacted with electrodes 812,electrode 812 may be used to split each droplet into two sub-droplets816, and sub-droplets 816 may be flowed into channel 810, as indicated.In an alternative embodiment, it will be appreciated that a flow may beestablished in channel 810 which causes sub-droplets 816 to flow in thesame direction, i.e., arrows B and C may indicate a flow in a commondirection, rather than opposite directions.

FIG. 9 illustrates a flow-through system 900 configured for adding beadsto droplets. System 900 includes channel 905 which intersects withchannel 910. A liquid filler fluid in channel 905 flows in the directionof arrow A. A liquid filler fluid in channel 910 flows in the directionof arrow B. Droplets 915 are provided in channel 905. The liquid fillerfluid in channel 905 is substantially immiscible with droplets 915.Droplets 915 flow through channel 905 into channel 910 at a velocitysufficient to cause them to impact a region on the wall of channel 910.The various dimensions of channel 905, channel 910 as it enters theintersection between the two channels, and channel 910 as it exits theintersection between the two channels, as well as the angle ofintersection and the velocity of filler fluid flow through therespective channels may be adjusted as needed to achieve thepre-selected droplet impact on the wall of channel 910. A magnet 920 isassociated with channel 910 at a position which is approximately thepoint of impact of droplets 915 on the wall of channel 910. The magnetmay be adjustable in order to align it with the appropriate location atwhich droplets 915 impact the wall of channel 910. The magnet may be anelectromagnet, which may be switched on and off. The magnet may be apermanent magnet, which is movable, e.g., generally in the direction ofthe axis indicated by arrow A. Beads 916 are provided in the fillerfluid which flows through channel 910. Beads 916 may be hydrophilic andmay be provided in a hydrophobic filler fluid. As each bead comes intoproximity with magnet 920, the bead is substantially immobilized onmagnet 920. When a droplet 915 impacts a bead 916 immobilized on magnet920, bead 916 is engulfed by the droplet, yielding bead containingdroplet 917. The bead-containing droplet 917 may continue to flowthrough channel 910 in the direction of arrow C. Various techniques maybe used to separate bead containing droplet 917 from the magnet 920 topermit bead containing droplet 917 to continue to flow-through channel910. For example, the surface tension of droplet 915 may be selected toovercome the attractive force of magnet 920 on the bead, as the beadcontaining droplet 917 is forced through channel 910 by the flowingfiller fluid. In this embodiment, it is not necessary to remove ordeactivate magnet 920. In another embodiment, magnet 920 is anelectromagnet, and the electromagnet is switched off to release thebead-containing droplet 917. In yet another embodiment, magnet 920 isremovable, and magnet 920 is physically moved away from channel 910 inorder to permit the release of bead containing droplet. The spacing ofdroplets 915 and beads 916 may be adjusted in order to achieve apre-selected number of beads and each droplet. For example, severalbeads may be permitted to collect at magnet 920 between each droplet 915in order to provide droplets with multiple beads. Droplets potentiallycontaining beads may be tested downstream, and sorted to exclude anydroplets which lack beads or which lack the pre-selected number ofbeads. Sorting may, for example, be based on optical properties and/orelectrical properties of the bead-containing droplets.

FIG. 10 illustrates a flow-through system 1000 which makes use ofdroplet operations to wash beads in droplets. Flow-through system 1000includes channel 1005, which intersects with channel 1010. A liquidfiller fluid in channel 1005 flows in the direction of arrow A. A liquidfiller fluid in channel 1010 flows in the direction of arrow B.Bead-containing droplets 1015 are provided in channel 1005. Washdroplets 1016 are provided in channel 1010. Wash droplets 1016 mayinclude a wash buffer. It will also be appreciated that in analternative embodiment, rather than washing the beads, the method isused to concentrate one or more substances onto the beads. In such otherembodiment, wash droplets 1016 may be replaced with sample droplets orother droplets including droplets including one or more targetsubstances for which the beads have affinity. In yet another embodiment,rather than a single magnet 1020 attracting bead-containing droplet 1015to the wall of channel 1010, one or more magnets may be provided aroundchannel 1010 and arranged to substantially immobilized the bead withinthe channel, but away from the wall of the channel. The size of channel1010 at magnet 1020 may be selected to ensure that wash droplets 1016impact immobilized bead containing droplet 1015 as they flow past magnet1020 or other magnet arrangement. The velocity of impact is selected tocause droplets 1016 to impact droplet 1015, merge with droplet 1015,followed by a breaking off of a new droplet 1017 moving in the directionof arrow C. In this manner, by sequentially merging the bead containingdroplet with a wash droplet in and breaking off a separate droplet, theliquid surrounding the bead-containing droplet maybe be depleted ofunwanted substances. Upon completion of the wash cycle, when thedepletion of unwanted substances is calculated to have been achievedbased on the number of wash droplets passed across the bead, the beadcontaining droplet may be released to continue to flow-through channel1010. Downstream, the bead containing droplets may be separated from theused wash droplets 1017. Thus, the invention provides a technique forwashing beads in a flow-through operation, wherein a bead containingdroplet is immobilized using a magnet, and one of more wash droplets arecaused to impact and merge with the bead-containing droplet, and whereinthe filler fluid flowing through the channel is at a velocity sufficientto cause one or more droplets to break off of the combined droplet,thereby leaving a bead containing droplet with a reduced amount of oneor more substances relative to the starting bead-containing droplet.Similarly, the invention provides a technique for concentrating asubstance on beads in a flow-through operation, wherein a beadcontaining droplet is immobilized using a magnet in a channel, and oneof more droplets including a target substance are caused to impact andmerge with the immobilized bead-containing droplet, thereby causing abead in the bead-containing droplet having affinity for the targetsubstance to concentrate target substance thereon. As with the washingoperation, the filler fluid flowing through the channel may cause one ormore droplets to break off of the combined droplet, thereby leaving abead containing droplet with an increased amount of one or moresubstances concentrated on the bead relative to the startingbead-containing droplet. The various sizes of channels 1005 and 1010, aswell as the angle of intersection between the two channels, may beadjusted in order to improve efficiency of the washing operation.Multiple beads may also be present in droplets 1015. The ratio ofspacing and velocity of bead containing droplets 1015 flowing throughchannel 1005 relative to the spacing and velocity of wash droplets orsample droplets flowing through channel 1010 may be adjusted to achievethe pre-selected effect. In yet another embodiment, channel 1010 mayinclude a series of sample droplets for concentrating sample onto theimmobilized bead, followed by a series of wash droplets for washing theimmobilized bead. In an alternative embodiment, the splitting off ofwash droplets following merging of the wash droplets with theimmobilized beat-containing droplet may be facilitated by dropletoperations mediated by electrodes, e.g. as described above withreference to FIG. 8.

In the various flow-through embodiments described herein, it is possiblefor droplets to be sorted to select out a pre-selected subset ofdroplets from the overall droplet population. For example, droplets maybe sorted as described in Link et al., US Patent Publication No.20080014518, entitled “Microfluidic Devices and Methods of Use Thereof,”published on Jan. 17, 2008, the entire disclosure of which isincorporated herein by reference for its teaching concerning sorting ofdroplets in microfluidic devices. Further, once droplets of interest areisolated, the droplets may be flowed onto a droplet actuator of theinvention for further analysis. For example, a subset of droplets ofinterest from a flow-through droplet sorting operation may be flowedinto a droplet operations gap of a droplet actuator where they aresubject to droplet operations mediated by electrodes. Similarly, asubset of droplets of interest from a flow-through droplet sortingoperation may be flowed into a reservoir of the droplet actuator, whichreservoir is coupled by a liquid path to a droplet operations gap of adroplet actuator, such that the droplets of interest may be transportedfrom the reservoir into the droplet operations gap where they may besubject to droplet operations mediated by electrodes. In one embodiment,multiple droplets of interest are pooled together in a reservoir of adroplet actuator prior to being subjected to droplet operations in adroplet operations gap of the droplet actuator.

Techniques Using Viscous, Solid, or Semi-Solid Samples

The invention provides droplet actuator devices, techniques and systemsfor making and using droplet actuators to process viscous, solid orsemi-solid samples. For example, the invention provides a technique forprocessing viscous, semisolid, and/or solid samples. Target substancesof interest are extracted from the viscous, solid or semi-solid sample,and then processed using standard droplet operations.

FIGS. 11A, 11B, and 11C illustrate a section of a droplet actuator 1100and a method of processing a viscous, solid or semi-solid sample on adroplet actuator. Droplet actuator 1100 includes a top substrate 1105and a bottom substrate 1110 separated by droplet operations gap 1112. Incertain embodiments, top substrate 1105 may be omitted. Dropletoperations electrodes 1115 (e.g., electrowetting electrodes) andreference electrodes (not shown) are associated with top substrate 1105and/or bottom substrate 1110. Droplet operations electrodes 1115 areconfigured for conducting one or more droplet operations in dropletoperations gap 1112. Top substrate 1105 includes an opening 1120 thereinfor loading sample 1125 into droplet operations gap 1112. Sample 1125includes one or more target substances 1130. As illustrated in FIG. 11A,droplet 1135 is positioned in droplet operations gap 1112 atop one ormore droplet operations electrodes 1115. FIG. 11B shows droplet 1135being transported into contact with sample 1125, such that one or moretarget substances 1130 is dissolved into droplet 1135. Transport ofdroplet 1135 may be effected using one or more droplet operations. Forexample, in one embodiment, transport is effected by sequentiallyactivating/deactivating electrodes 1115. Droplet 1135 may be transportedaway from sample 1125 via droplet operations as shown in 11C. Droplet1135 that potentially included one or more target substances may be usedas input for conducting one or more assays to identify and/or quantifyone or more target substances 1130. In one embodiment, sample 1125 issufficiently viscous, semi-solid, or solid in order to permit droplet1135 to contact sample 1125 and be transported away from sample 1125without being substantially mixed with sample 1125.

FIGS. 11A, 11B, and 11C illustrate a general principle in which a microor nano liquid is transported into contact with a viscous or solidsample for collection of a target substance and then transported away.As illustrated, using one or more droplet operations, droplet 1135contacts sample 1125, which brings droplet 1135 into lateral contactwith sample 1125. However, it will be appreciated that sample 1125 maybe positioned at any angle relative to droplet 1135, e.g., above orbelow droplet 1135. For example, sample 1125 may project only slightlyinto droplet operations gap 1112, in which case, droplet 1135 may betransported along a path of electrodes underneath sample 1125. In thisexample, contact is between the top of droplet 1135 and the bottom ofsample 1125. Alternatively, sample 1125 may be exposed to dropletoperations gap 1112 and droplet 1135 via an opening (not shown) inbottom substrate 1110.

The methods of the invention are particularly suitable for testsinvolving viscous, solid or semi-solid samples. Samples may, forexample, be environmental samples, process samples, or biologicalsamples. Examples of suitable samples include sputum, coagulated blood,animal tissue samples, plant tissue samples, soil samples, rock samples,and the like. In some cases, samples are sufficiently viscous,semi-solid or solid to permit a droplet to contact the sample and betransported away from the sample without being substantially mixed withthe sample. Further, the sample may include foreign matter, such as amatrix (e.g., a swab) used to collect the sample. For example, when adroplet of sputum is loaded, it may not be readily transportable usingdroplet operations then a droplet that lyses sputum can be brought incontact with sputum. After incubation and preferably some agitation ofthe lysis droplet, the sputum will be liquefied rendering it to betransportable using droplet operations.

Droplet 1135 may be aqueous or non-aqueous. In one embodiment, droplet1135 is an aqueous buffer established at a pH which is suitable fordissolving sample 1125. Droplet 1135 may also include one or morereagents. The chemical characteristics of droplet 1135 may be adjustedto render droplet 1135 suitable for acquiring one or more targetsubstances 1130. In one example, droplet 1135 includes a lysis buffersolution. A lysis buffer solution is used to lyse cells for use inassays involving target substances, which are sub-components of thecells. In some embodiments, droplet 1135 includes one or more beads,e.g., magnetically responsive or non-magnetically responsive beads.Examples of suitable magnetically responsive beads are described in U.S.Pat. No. 7,205,160, entitled, “Multiplex flow assays preferably withmagnetic particles as solid phase,” granted on Apr. 17, 2007. The beadsmay have an affinity for one or more target substances or contaminants.For example, the beads may have affinity for target cells, protein, DNA,and/or antigens. In one example, the beads may have an affinity for oneor more target substances 1130 from the sample 1125 of interest.

Examples of droplet actuator techniques for immobilizing magnetic beadsand/or non-magnetic beads are described in the foregoing internationalpatent applications and in Sista, et al., U.S. Patent Application Nos.60/900,653, entitled “Immobilization of Magnetically-responsive BeadsDuring Droplet Operations,” filed on Feb. 9, 2007; Sista et al., U.S.Patent Application No. 60/969,736, entitled “Droplet Actuator AssayImprovements,” filed on Sep. 4, 2007; and Allen et al., U.S. PatentApplication No. 60/957,717, entitled “Bead Washing Using PhysicalBarriers,” filed on Aug. 24, 2007; the entire disclosures of which areincorporated herein by reference.

Gel Electrophoresis Techniques

The invention provides droplet actuator devices including a gel for usein gel electrophoresis, along with techniques and systems for conductinggel electrophoresis on a droplet actuator. The gel electrophoresistechniques of the invention are useful for separating substances presentin a droplet on a droplet actuator. For example, the invention is usefulfor separating complex biomolecules (e.g., proteins and/or nucleicacids) using an electric current applied to a gel matrix on a dropletactuator. The gel matrix may, for example, be a cross-linked polymerwhose composition and porosity are selected based on the specific weight(e.g., molecular weight) and composition of the substances beinganalyzed. In one embodiment, the gel may be composed of differentconcentrations of acrylamide and a cross-linker to producedifferent-sized mesh networks of polyacrylamide. Polyacrylamide may beused to separate and analyze proteins or small nucleic acids (e.g., DNA,RNA, or oligonucleotides). In another embodiment, the gel may becomposed of a purified agarose matrix. Agarose gels may be used toseparate larger nucleic acids and/or complex biomolecules.

The methods of the invention make use of gel electrophoresis on adroplet actuator for analytical purposes (e.g., separation andquantitation of a specific target(s)). In another embodiment, gelelectrophoresis may be used as a preparative technique (e.g., forisolation of a specific target(s)) prior to use of other assaytechniques for further characterization of a substance. Other assaytechniques may, for example, include PCR, cloning, nucleic acidsequencing, immunoassays, enzymatic assays, exposure to sensors, etc. Inanother embodiment, a “capture” droplet may be used to capture a targetdroplet as it elutes off the gel slug as a fraction collector, e.g.using the techniques described with reference to FIG. 11.

FIGS. 12A, 12B, and 12C illustrate a section of droplet actuator 1200and a process of separating and analyzing a sample using gelelectrophoresis. Droplet actuator 1200 may include bottom substrate 1210separated from top substrate 1214 by droplet operations gap 1213. Path1216 of droplet operations electrodes 1217 is arranged on bottomsubstrate 1210; however, it will be appreciated that droplet operationselectrodes and/or ground electrodes may be associated with top substrate1214 and/or bottom substrate 1210. Droplet operations electrodes 1217may, for example, be electrowetting electrodes. Electrophoresiselectrodes 1218 a and 1218 b, are arranged on top substrate 1214, butmay be on either or both substrates. One of electrodes 1218 a and 1218 bmay be a negative electrode, while the other may be a positiveelectrode.

Droplet operations gap 1213 may be provided with one or more geldroplets 1226 and one or more catalyst droplets 1230, although in somecases neither a catalyst nor a catalyst droplet are required. In somecases, the catalyst may just be photoinitiation. Gel droplets 1226 maytypically be from about 1× to about 5× or larger droplets. A 3× droplet,for example, has a footprint that is approximately 3 times the area ofone droplet operations electrode 1217. Gel droplet 1226 may, forexample, include reagents suitable for forming a polyacrylamide gel,such as acrylamide, bis-acrylamide, and buffer. Gel droplet 1226 remainsin a liquid form until polymerization of the acrylamide is initiated bythe addition of a catalyst. Catalyst droplet 1230 contains the chemicalreagents required to accelerate polymerization of gel droplet 1226. Forexample, catalyst droplet 1230 may includeN,N,N,N-Tetramethyl-Ethylenediamine (TEMED) and ammonium persulfate toaccelerate polymerization of the acrylamide in gel droplet 1226.

Droplet operations gap 1213 may be provided with one or more sampledroplets, e.g., sample droplet 1234. Sample droplet 1234 includes one ormore target substances 1242 to be evaluated. Target substances 1242 may,for example, be fluorescently labeled proteins or nucleic acids. Toevaluate target substances 1242, an imaging device 1240 is associatedwith droplet actuator 1200. Imaging device 1240 may be used to capturedigital images of substances separated in gel droplet 1226, such aslabeled proteins or nucleic acids. In some cases, imaging device 1240may capture images through top substrate 1214, which may be, forexample, a glass or a plastic plate that is substantially transparent.

FIG. 12A shows a first step in which droplet operations may be executedin order to form a gel for conducting gel electrophoresis on dropletactuator 1200. Activated electrodes are shown in black. Using one ormore droplet operations, gel droplet 1226 may be elongated along severaldroplet operations electrodes in contact with electrophoresis electrodes1218 a and 1218 b. Catalyst droplet 1230 may be transported using one ormore droplet operations into contact with gel droplet 1226. Catalystdroplet 1230 and gel droplet 1226 merge, initiating polymerization ingel droplet 1226 to form the gel matrix for electrophoresis. FIG. 12Bshows a second step in which sample droplet 1234 is transported intocontact with the polymerized gel droplet 1226. FIG. 12C shows a thirdstep in which an electrical potential (e.g., about 40 to about 100volts) may be applied to gel droplet 1226 via electrophoresis electrodes1218 a and 1218 b. In some cases, electrode 1218 a might directlycontact droplet 1234. The electrical current causes target substances1242 in sample droplet 1234 to electrophorese into and through geldroplet 1226. Separation of target substances 1242 in gel droplet 1226is typically determined by charge such that different molecules willmove at different rates. As an example, target substances 1242 may benegatively charged (e.g., nucleic acids) and migrate fromelectrophoresis electrode 1218 a (i.e., negative electrode) towardelectrophoresis electrode 1218 b (i.e., positive electrode). Imagingdevice 1240 may be used to capture an image of separated targetsubstances 1242 in gel droplet 1226 and/or as they elute off the end ofgel droplet 1226. The captured image may be used to identify and/orquantitate different target substances 1242 in sample 1234.

It will be appreciated that the method of the invention also provides ageneric method of forming a polymerized structure in a dropletoperations gap of a droplet actuator. The method may include using oneor more droplet operations to form a first droplet into a pre-selectedshape, and to contact the first droplet with a second droplet to causepolymerization of the combined droplets. One of the first droplet and/orsecond droplet may be a polymer droplet, while the other of the firstdroplet and/or second droplet may be a catalyst droplet. In addition touse for forming gels for electrophoresis, the method may be used toprovide a physical obstacle on a droplet actuator. The physical obstaclemay, for example, be useful for sealing off a region of the dropletactuator. In one embodiment a droplet actuator is provided that includesa barrier in the droplet operations gap establishing two regions on thedroplet actuator. An opening is provided in the barrier, and electrodesare arranged for transporting droplets through the opening. When it isdesirable to close the opening, a polymer droplet is polymerized in theopening. For example, a polymer droplet may be transported into theopening. A catalyst droplet may be combined with the polymer droplet inthe opening. Upon polymerization, the opening may be substantiallyclosed.

Fluidics System for Loading Droplet Actuator

The invention provides a fluidics system and technique for using thesystem for loading liquids onto a droplet actuator. The invention alsoprovides droplet actuators loaded using the fluidics system and methodof the invention and methods of using such droplet actuators to conductdroplet operations. In some embodiments, the loading provides a dropletactuator in which the droplet operations gap or a reagent storagechannel is fully filled with filler fluid and reagents that issubstantially lacking in air bubbles.

FIG. 13A-13I are schematic diagrams of fluidics system 1300 for loadingliquid receptacle, such as a channel or droplet operations gap of adroplet actuator, with liquid. Fluidics system 1300 may include anarrangement of one or more valves, one or more pumps, one or morecapillaries, and one or more liquid supply vessels; all fluidlyconnected. Additionally, a droplet actuator may be fluidly connected tofluidics system 1300, such that liquid may be flowed from fluidicssystem 1300 into a liquid receptacle of droplet actuator 1320.

Fluidics system 1300 includes a plurality of valves, illustrated here aspinch valves (PV): PV1, PV2, PV3, PV4, and PV5. A pinch valve is a valvein which a flexible tube is pinched between one or two moving externalelements in order to stop the flow through the tube.

Fluidics system 1300 includes one or more pumps, illustrated here asperistaltic pump P1. In a peristaltic pump, liquid is contained within aflexible tube fitted inside a circular pump casing. A rotor with one ormore of rollers, shoes, or wipers that are attached to the externalcircumference compresses the flexible tube. As the rotor turns, the partof tube under compression closes, which forces the liquid to be pumpedto move through the tube. Referring to FIG. 13A, peristaltic pump P1 maycontrolled to operate in a clockwise (CW) and counter clockwise (CCW)direction. The peristaltic pump may be replaced with any suitable pumptype, such as gear pumps, progressing cavity pumps, roots-type pumps,reciprocating-type pumps, double-diaphragm pumps, peristaltic pumps,kinetic pumps, centrifugal pumps, eductor-jet pumps, etc.

Fluidics system 1300 also includes a pump, such as syringe pump P2.Syringe pump includes a cylinder that holds a quantity of liquid, suchas filler fluid (e.g., silicone oil), which is expelled by a piston. Thepiston may be advanced or retracted by a motor (not shown) connectedthereto, in order to provide smooth pulseless flow.

Fluidics system 1300 includes a liquid supply vessel V1, which is, forexample, any vessel for holding a quantity of liquid, such as fillerfluid (e.g., silicone oil).

Fluidics system 1300 includes another liquid supply, illustrated here asa multi-well plate (MWP1). MWP1 contains, for example, multiplereservoirs including reagents 1310 (and/or sample) under a layer offiller fluid 1314 (e.g., silicone oil). A mechanically or roboticallycontrolled supply line 1318 may be manipulated in the X, Y, and Zdirections in order to access a certain one of the multiple fluids thatare contained in MWP1. In an alternative embodiment, multiple supplylines may be provided extending into the MWP1 reservoirs from the top,or through an opening in the reservoirs, such as opening in the bottomof the reservoirs.

Fluidics system 1300 includes a capillary CP1, which is a small diametertube of any pre-selected length, depending on the pre-selected quantityof liquid to be contained therein. Various liquid lines L fluidlyconnect the parts of the invention.

Fluidics system 1300 may include droplet actuator 1320, which is thedroplet actuator to be loaded by fluidics system 1300. Droplet actuator1320 is fluidly connected to fluidics system 1300 via one or more liquidinput/output ports. The ports provide a fluid path from an exterior ofthe droplet actuator into a droplet operations gap of the dropletactuator or into another reservoir in the droplet actuator, such as achannel reservoir. In one example, the droplet operations gap of dropletactuator 1320 is fluidly connected to fluidics system 1300 via ports C1,C2, and C3. In some cases, the ports may provide access to one or morechannels within droplet actuator 1320, and the one or more channels areused to supply filler fluids and/or reagents into a droplet operationsgap of droplet actuator 1320.

Referring again to FIG. 13A, the elements of fluidics system 1300 arefluidly connected as follows. A liquid line L fluidly connects vessel V1to one opening of valve PV1. A liquid line L fluidly connects to theopposite opening of valve PV1 to an opening of T-connection T1. A liquidline L fluidly connects a first branch of T1 to an opening of valve PV2.A liquid line L fluidly connects the opposite opening of valve PV2 toport C1 of droplet actuator 1320. A liquid line L fluidly connects asecond branch of T1 to one opening of peristaltic pump P1. A liquid lineL fluidly connects to the opposite opening of peristaltic pump P1 to oneopening of capillary CP1. A liquid line L fluidly connects the oppositeopening of capillary CP1 to a T-connection, T2. A liquid line L fluidlyconnects a first branch of T2 to one opening of valve PV4. A liquid lineL fluidly connects the opposite opening of valve PV4 to port C2 ofdroplet actuator 1320. A liquid line L fluidly connects a second branchof T2 to one opening of valve PV5. A liquid line L fluidly connects theopposite opening of valve PV5 to supply line 1318 that fluidly connectsto MWP1. An input/output port of syringe pump P2 fluidly connects toport C3 of droplet actuator 1320 through valve PV3. Note that all liquidlines of fluidics system 1300 may be capillaries and that capillary CP1may be formed of an extended length of capillary that couplesperistaltic pump P1 and junction T2.

Fluidics system 1300 of FIG. 13A is exemplary only, other systemvariations are possible. For example, syringe pump P2 may be replacedwith other types of pumps. Alternatively, fluidics system 1300 mayinclude a single pump only. An exemplary method of purging air fromfluidics system 1300 and droplet actuator 1320 and filling fluidicssystem 1300 and droplet actuator 1320 with substantially bubble-freeliquid is described with reference to FIGS. 13B-13I.

Purging Fluidics System—Step 1

FIG. 13B, with reference to Table 1 below, illustrates a purging step inwhich valves PV1 and PV5 are open, valves PV2, PV3, and PV4 are closed,peristaltic pump P1 is activated in the CW direction, and syringe pumpP2 is stopped. This arrangement establishes a flow of liquid from vesselV1, through valve PV1, through peristaltic pump P1, and into capillaryCP1. Liquid displaces air in the path from vessel V1 to capillary CP1.Air is vented through valve PV5 to supply line 1318. Upon completion ofthis step, a quantity of liquid from vessel V1 that is sufficient tofill the liquid line between T1 and port C1 of droplet actuator 1320 iscontained in capillary CP1.

TABLE 1 PV1 PV2 PV3 PV4 PV5 P1 P2 OPEN CLOSED CLOSED CLOSED OPEN CW STOP

Purging Fluidics System—Step 2

FIG. 13C, with reference to Table 2 below, illustrates a second purgingstep in which valves PV2 and PV4 are open, valves PV1, PV3, and PV5 areclosed, peristaltic pump P1 is activated in the CCW direction, andsyringe pump P2 is stopped. This arrangement establishes a flow ofliquid from capillary CP1, through peristaltic pump P1, through valvePV2, and into port C1 of droplet actuator 1320. Liquid displaces air inthe path from T1 to port C1 of droplet actuator 1320. Air is ventedthrough port C2 of droplet actuator 1320 and through valve PV4.

TABLE 2 PV1 PV2 PV3 PV4 PV5 P1 P2 CLOSED OPEN CLOSED OPEN CLOSED CCWSTOP

Purging Fluidics System—Step 3

FIG. 13D, with reference to Table 3 below, illustrates a third purgingstep in which valves PV1 and PV5 are open, valves PV2, PV3, and PV4 areclosed, peristaltic pump P1 is activated in the CW direction, andsyringe pump P2 is stopped. This arrangement establishes a flow ofliquid from vessel V1, through valve PV1, through peristaltic pump P1,through capillary CP1, through one branch of T2, through valve PV5, andthrough supply line 1318 to MWP1. Liquid displaces air in the path fromvessel V1 to MWP1. Air is vented through supply line 1318 to MWP1.

TABLE 3 PV1 PV2 PV3 PV4 PV5 P1 P2 OPEN CLOSED CLOSED CLOSED OPEN CW STOP

Purging Fluidics System—Step 4

FIG. 13E, with reference to Table 4 below, illustrates a fourth purgingstep in which valves PV1, PV2, and PV3 are open, valves PV4 and PV5 areclosed, peristaltic pump P1 is stopped (i.e., pump P1 acts as a closedvalve), and syringe pump P2 is activated in a direction selected to pullliquid from fluidics system 1300. This arrangement establishes a flow ofliquid from vessel V1, through valve PV1, through T1, through valve PV2,through droplet actuator 1320 from port C1 to port C3, through valvePV3, and into the cylinder of syringe pump P2. Liquid displaces air inthe path from vessel V1 to syringe pump P2. Droplet actuator 1320 ispurged of air. Air is drawn into syringe pump P2.

TABLE 4 PV1 PV2 PV3 PV4 PV5 P1 P2 OPEN OPEN OPEN CLOSED CLOSED STOP PULL

Purging Fluidics System—Step 5

FIG. 13F, with reference to Table 5 below, illustrates a fifth purgingstep in which valves PV3, PV4, and PV5 are open, valves PV1 and PV2 areclosed, peristaltic pump P1 is stopped (i.e., pump P1 acts as a closedvalve), and syringe pump P2 is activated in a direction to push liquidinto fluidics system 1300. This arrangement establishes a flow of liquidfrom syringe pump P2, through valve PV3, through droplet actuator 1320from port C3 to port C2, through valve PV4, through T2, through valvePV5, and through supply line 1318 to MWP1. Liquid displaces air in thepath from syringe pump P2 to MWP1. Air is vented through supply line1318 to MWP1.

TABLE 5 PV1 PV2 PV3 PV4 PV5 P1 P2 CLOSED CLOSED OPEN OPEN OPEN STOP PUSH

At the completion of this step, all air has been purged from fluidicssystem 1300, and droplet actuator 1320. All liquid lines and elements offluidics system 1300 and all channels of droplet actuator 1320 arefilled with liquid and substantially free of air bubbles.

Loading Droplet Actuator—Step 1

FIG. 13G, with reference to Table 6 below, illustrates an exemplaryfirst step in a method of loading a droplet actuator. Fluidics system1300 has two pumps, peristaltic pump P1 and syringe pump P2, that areavailable for loading reagents into droplet actuator 1320. Peristalticpump P1 of fluidics system 1300 is used for loading reagents intodroplet actuator 1320. Valves PV1 and PV5 are open, valves PV2, PV3, andPV4 are closed, peristaltic pump P1 is activated in the CCW direction,and syringe pump P2 is stopped. Additionally, using the xyz-motion,supply line 1318 is inserted into a well of MWP1 that contains thepre-selected reagent 1310. A certain amount of reagent 1310 is drawnfrom MWP1 in a flow loop through peristaltic pump P1 and toward vesselV1, as indicated in FIG. 13G. Subsequently, supply line 1318 is liftedout of reagent 1310 and into filler fluid 1314 and a certain amount offiller fluid 1314 is drawn from MWP1. In some embodiments, supply line1318 may oscillate up and down in the well to create multiple slugs. Atrain of reagent slugs that are separated by filler fluid flows towardcapillary CP1. When the entire train of reagent slugs is present withinCP1, peristaltic pump P1 is stopped.

TABLE 6 PV1 PV2 PV3 PV4 PV5 P1 P2 OPEN CLOSED CLOSED CLOSED OPEN CCWSTOP

Loading Droplet Actuator—Step 2

FIG. 13H, with reference to Table 7 below, illustrates an exemplary nextstep in a method of loading a droplet actuator. Fluidics system 1300 hastwo pumps, peristaltic pump P1 and syringe pump P2, that are availablefor loading reagents into droplet actuator 1320. Peristaltic pump P1 offluidics system 1300 is used for loading reagents into droplet actuator1320. Valves PV2 and PV4 are open, valves PV1, PV3, and PV5 are closed,peristaltic pump P1 is activated in the CW direction, and syringe pumpP2 is stopped. This arrangement establishes a flow loop through dropletactuator 1320 that includes peristaltic pump P1 and capillary CP1, asindicated in FIG. 13H. The train of reagent slugs within capillary CP1flows into droplet actuator 1320, from port C2 toward port C1, anddroplet actuator 1320 is, thus, loaded with the pre-selected reagent andready for operation.

TABLE 7 PV1 PV2 PV3 PV4 PV5 P1 P2 CLOSED OPEN CLOSED OPEN CLOSED CW STOP

Direct Dispensing Method of Loading a Droplet Actuator

FIG. 13I, with reference to Table 8 below illustrates another step in amethod of loading a droplet actuator. Fluidics system 1300 has twopumps, peristaltic pump P1 and syringe pump P2, that are available forloading reagents into droplet actuator 1320. Syringe pump P2 of fluidicssystem 100 is used for loading reagents into droplet actuator 1320.Valves PV3, PV4, and PV5 are open, valve PV1 is closed, PV2 isoptionally closed, peristaltic pump P1 is stopped (i.e., pump P1 acts asa closed valve), and syringe pump P2 is activated in a direction to pullliquid from fluidics system 1300. Additionally, using the xyz motion,supply line 1318 is inserted into a pre-selected well of MWP1 thatcontains the pre-selected reagent 1310. A certain amount of reagent 1310is drawn from MWP1 in a flow loop through droplet actuator 1320 fromport C2 to port C3 and toward syringe pump P2, as indicated in FIG. 13I.In one example, syringe pump P2 is used for loading a large volumereagent slug from MWP1 into droplet actuator 1320.

TABLE 8 PV1 PV2 PV3 PV4 PV5 P1 P2 CLOSED CLOSED OPEN OPEN OPEN STOP PULL

FIGS. 14A-14F are schematic diagrams of an example of another fluidicssystem 1400 for loading a droplet actuator with liquid. With referenceto FIG. 14A, fluidics system 1400 may include any arrangement of one ormore valves, one or more pumps, one or more capillaries, and one or moreliquid supply vessels; all fluidly connected. A droplet actuator to beloaded is fluidly connected to fluidics system 1400. In one example,fluidics system 1400 is substantially the same as fluidics system 1300,except that a vent path that includes a pinch valve PV6 is providedbetween peristaltic pump P1 and capillary CP1, and MWP1 is replaced witha capillary CP2, which is preloaded with a certain train of reagentslugs. Note that, like fluidics system 1300, all liquid lines L offluidics system 1400 may be capillaries or other tubes and thatcapillary CP1 may be an extended length of capillary that couplesperistaltic pump P1 and junction T2. Similarly, capillary CP2 may beformed of an extended length of capillary coupled to pinch valve P5.

Fluidics system 1400 is exemplary only, other system variations arepossible within the scope of the invention. For example, syringe pump P2may be replaced with other types of pumps. Alternatively, fluidicssystem 1400 may include a single pump only.

Purging Air from Fluidics System—Step 1

FIG. 14B, with reference to Table 9 below, illustrates a first purgingstep, in which valve PV1 is optionally open, valves PV3 and PV4 areopen, valves PV2, PV5, and PV6 are closed, peristaltic pump P1 isactivated in the CW direction, and syringe pump P2 is activated in adirection to pull liquid from fluidics system 1400. By using peristalticpump P1 and syringe pump P2 simultaneously, liquid is drawn from vesselV1, through valve PV1, through peristaltic pump P1, through capillaryCP1, through valve PV4, through droplet actuator 1420, through valvePV3, and into syringe pump P2. Liquid displaces air in the path fromvessel V1 to syringe pump P2. Air is drawn into syringe pump P2.

TABLE 9 PV1 PV2 PV3 PV4 PV5 PV6 P1 P2 OPEN CLOSED OPEN OPEN CLOSEDCLOSED CW PULLPurging air from Fluidics System—Step 2

FIG. 14C, with reference to Table 10 below, illustrates a second purgingstep, in which valves PV1, PV2, PV3, PV5, and PV6 are open, valve PV4 isclosed, peristaltic pump P1 is stopped (i.e., pump P1 acts as a closedvalve), and syringe pump P2 is activated in a direction to push liquidinto fluidics system 1400. This arrangement establishes a flow of liquidfrom syringe pump P2, through droplet actuator 1420 from port C3 to portC1, through valve PV2, through T1, through valve PV1, and into vesselV1, as indicated in FIG. 14C. Liquid displaces air in the path fromsyringe pump P2 to vessel V1. Air is vented at vessel V1.

TABLE 10 PV1 PV2 PV3 PV4 PV5 PV6 P1 P2 OPEN OPEN OPEN CLOSED OPEN OPENSTOP PUSHPurging Air from Fluidics System—Step 3

FIG. 14D, with reference to Table 11 below, illustrates a third purgingstep, in which valves PV1 and PV5 are open, valves PV2, PV3, PV4, andPV6 are closed, peristaltic pump P1 is activated in the CCW direction,and syringe pump P2 is stopped. Peristaltic pump P1 pumps liquid fromcapillary CP2, through valve PV5, and through T2. Peristaltic pump P1 isoperated until such time that any air that precedes the train of reagentslugs from preloaded capillary CP2 is trapped between peristaltic pumpP1 and T3.

TABLE 11 PV1 PV2 PV3 PV4 PV5 PV6 P1 P2 OPEN CLOSED CLOSED CLOSED OPENCLOSED CCW STOPPurging Air from Fluidics System—Step 4

FIG. 14E, with reference to Table 12 below, illustrates a fourth purgingstep, in which valves PV1 and PV6 are open, valves PV2, PV3, PV4, andPV5 are closed, peristaltic pump P1 is activated in the CW direction,and syringe pump P2 is stopped. This arrangement establishes a flow ofliquid between vessel V1 and valve PV6, which is the vent path. Pump P1pushes air trapped between peristaltic pump P1 and T3 through PV6,through which air is vented from fluidics system 1400.

TABLE 12 PV1 PV2 PV3 PV4 PV5 PV6 P1 P2 OPEN CLOSED CLOSED CLOSED CLOSEDOPEN CW STOP

At the completion of this step, all air has been purged from fluidicssystem 1400 and droplet actuator 1420, as all liquid lines and elementsof fluidics system 1400 and all channels of droplet actuator 1420 arefilled with liquid and substantially free of air bubbles.

Loading a Droplet Actuator

FIG. 14F, with reference to Table 13 below illustrates a step in amethod of loading a droplet actuator. Valves PV2 and PV4 are open, andvalves PV1, PV3, PV5, and PV6 are closed, peristaltic pump P1 isactivated in the CW direction, and syringe pump P2 is stopped. Becausefluidics system 1400 contains all filler fluid and reagent slugsnecessary for the operation of droplet actuator 1420, the pumping actionof peristaltic pump P1 moves the train of reagent slugs into dropletactuator 1420, from port C2 to port C1, as indicated in FIG. 14F.

TABLE 13 PV1 PV2 PV3 PV4 PV5 PV6 P1 P2 CLOSED OPEN CLOSED OPEN CLOSEDCLOSED CW STOP

Sample Processing

The invention provides a droplet actuator device and methods forprocessing samples for use on a droplet actuator device. For example,the invention provides methods of processing samples for conductinggenetic analysis of microbiological organisms in a biological sample.The device and methods of the invention may be used to detect andidentify microorganisms such as bacteria, viruses, and/or fungi in abiological sample. Examples of biological samples include, blood,plasma, serum, isolated microorganisms, nucleic acid spiked into anassay buffer, other samples described herein, and other known sampletypes. In various embodiments, the invention provides for dropletactuator-based sample preparation and nucleic acid analysis. The deviceand methods of the invention may, in one embodiment, be used for rapidand accurate identification of atypical bacteria that have specifictreatment implications, such as selection of effective antibiotics andlength of therapy. For example, in the immunosuppressed population theability to distinguish between bacteria, viruses, and fungi both rapidlyand accurately will be life-saving.

Sample Preprocessing

The invention provides a droplet actuator device and methods forpre-processing samples prior to introduction of the samples onto adroplet actuator. Prior to transfer of sample to the droplet actuator,the sample may be combined with magnetic beads having affinity foranalytes (e.g., DNA and/or RNA) of interest. The analytes of interestmay be bound to the magnetically responsive capture beads. Themagnetically responsive beads may be concentrated in a small part of theprocessed sample volume. The reduced sample volume that contains themagnetically responsive beads may be loaded onto the droplet actuator.For example, volume reduction may be from about ≧1 milliliter (mL) toabout ≦10 microliters (μL).

The droplet actuator may be provided as part of a system which isprogrammed to execute analysis protocols using electrical fields toperform droplet operations. For example, in a real-time PCR assay,thermocycling is accomplished by transporting reaction droplets throughisothermal temperature zones within the droplet actuator rather than bycycling the heaters (“flow-through” PCR). This and other PCR approachesare described in Pollack et al., International Patent Application No.PCT/US 06/47486, entitled “Droplet-Based Biochemistry,” filed on Dec.11, 2006, the entire disclosure of which is incorporated herein byreference.

The droplet actuator may be electrically coupled with the system usingmating alignment features to ensure proper positioning. The matingalignment features align the droplet actuator with various functionalelements, such as heaters, magnets, and detection elements, that arealigned with specific regions of droplet actuator. A sample is loadedinto the sample well. The sample well may be sealed before the analysisprotocol can be started. Once the analysis protocol is started, itproceeds to completion without requiring operator intervention. Usingone or more droplet operations, the sample is combined in the cassettewith appropriate reagents, such as lysis buffer, capture buffer, andcapture beads, as required by the analysis protocol. Meanwhile thedroplet actuator is primed for performing the final assay (e.g.,real-time PCR).

The low thermal mass of the droplets combined with the speed and agilitywith which they can be positioned using one or more droplet operationsenables extremely rapid and precise thermal profiles to be achieved. Theinventors have successfully implemented real-time PCR in microfluidicformat, which includes tests for bacterial and fungal pathogens(Bacillus anthracis, Franciscella tularensis, Candida albicans,Mycoplasma pneumoniae, Eschericia coli, Methicillin-resistantStaphylococcus aureus (MRSA)), human gene targets (RPL4, CFTR, PCNA) andRNA.

FIG. 15A shows a plot of real-time PCR data for detection of MRSA usingdigital microfluidics. FIG. 15B shows a plot of real-time PCR data fordetection of Bacillus anthracis using digital microfluidics.

Referring to FIG. 15A, for detection of MRSA by real-time PCR, a forwardprimer mecii574 (5′-GTC AAA AAT CAT GAA CCT CAT TAC TTA TG-3′) andreverse primer Xsau325 (5′-GGA TCA AAC GGC CTG CAC A-3′) were used toamplify a 176 bp fragment of Staphylococcus aureus genomic DNA (ATCC#700699D-5). The 50 μl PCR mix was comprised of 20 mM Tris HCl (pH 8.4),50 mM KCl, 200 μM dNTPs, 1 μM of each primer, 2× Evagreen (Biotium),6.125U of KAPA2G Fast DNA polymerase (Kapa Biosystems). This mix wasadjusted to 50 μl with H20 and approximately 1-2 μL of this mixture wasloaded in one of the droplet actuator reservoirs.

The protocol performed on the droplet actuator was to dispense two (450nL) droplets from the reservoir and combine them to form a single (900nL) reaction droplet. When sample and reagent are provided separatelyone droplet would be for the sample and the other droplet would be forthe 2× reaction mixture. The droplets are then transported to the 95° C.zone and, following an initial activation step, the droplets are cycledbetween the 60° C. and 95° C. zones 40 times. A fluorescence reading wastaken at the end of each extension cycle within the 60° C. zone. The twopositions were spanned by 16 electrodes and the droplets were typicallytransferred at a rate of 20 electrodes per second, thus the time totransfer the droplet between the two thermal zones was approximately 750milliseconds (ms). Real-time PCR curves obtained for 10-fold dilutionsof MRSA genomic DNA concentration exhibited roughly the expected 3.3cycle separation. The results were confirmed by gel analysis of theamplified product collected from the droplet actuator (not shown). Inall cases the amplified product was of the expected length and noby-products were observed.

Referring to FIG. 15B, an experiment was also conducted to evaluatedetection of Bacillus anthracis (anthrax) using digital microfluidicPCR. These experiments were performed using an early version of adroplet actuator and instrument and were not optimized for speed.Genomic DNA (chromosomal & plasmids) and primers targeted against B.anthracis protective antigen were provided from a commercially availablekit (Idaho Technology, Salt Lake City, Utah) and combined with a similarreaction mixture to that described above for detection of MRSA. Theseexperiments were performed with varying amounts of the DNA (i.e., 1 ng,100 pg, 10 pg, 1 pg genomic DNA) added into the reactions which wereamplified on the droplet actuator. Cycling conditions were 10 sec at 95°C. and 60 sec at 60° C. times 40 cycles. The data demonstrate theexpected quantitation with detection down to 1 pg of genomic DNA.

Capture, Concentration and Elution of Nucleic Acids

The invention provides droplet actuator devices, techniques and systemsfor capturing, concentrating and/or eluting nucleic acids.

FIG. 16 shows a plot resulting from amplification of MRSA genomic DNAcaptured, concentrated and eluted on a droplet actuator. In operation, adroplet actuator is electrically coupled to the instrument (not shown).A suspension of magnetically responsive beads that contain captured DNAin a lysis solution was loaded into a sample reservoir of the dropletactuator. In an alternative embodiment, the lysis solution that containsMRSA genomic DNA may be provided as a droplet on a droplet actuator andcombined with the bead-containing droplet on the droplet actuator. Apermanent magnet located in close proximity to the droplet actuator isused to collect the magnetically responsive beads at the bottom of thewell. A single droplet is dispensed from the sample reservoir. Thesingle droplet contains substantially all of the magnetically responsivebeads from the original sample, effectively concentrating the beads by afactor of about 50 or more. The droplet is then transported to a washstation where the magnetically responsive beads are magneticallyimmobilized and repeatedly washed. For about the last several washes,the wash fluid is switched to an elution buffer. The droplets thatcontain eluted DNA are accumulated within another reservoir. Thepurified DNA droplet is subsequently dispensed from the reservoir andmixed with multiple sets of PCR reagent droplets. The droplets aretransported to the heater zone of the deck and flow-through real-timePCR is performed.

As a proof of concept, genomic MRSA DNA was added to several mL of celllysis solution that contained DNA-capture magnetically responsive beads.The beads were then concentrated off-actuator and transferred in 15 μLof solution to the sample well of the droplet actuator. The beads werefurther concentrated into a single (˜300 nL) DNA capture droplet. TheDNA capture droplet was washed using a merge-and-split protocol with 8droplets of TE buffer (pH 7.0) and then eluted with 12 droplets of TEbuffer (pH 8.5) into a reservoir. Droplets of purified DNA were thendispensed and mixed in a 1:1 ratio with a real-time PCR mix.

Data indicate that sample concentration, elution, and detection weresuccessfully performed on a droplet actuator.

Sample Preparation on a Droplet Actuator

On-actuator preparation of biological samples provides a method forsensitive isolation of nucleic acids using one or more dropletoperations to perform separation protocols. Droplet actuator-basedsample preparation includes lysis (when necessary) of a sample, captureof nucleic acids (e.g., on magnetically responsive beads),pre-concentration of nucleic acids, a washing of captured nucleic acidsto remove unbound material prior to analysis. The flexibility andprogrammability of the droplet actuator provides for variation in theorder in which sample and reagents may be combined during samplepreparation.

In one embodiment, a sample droplet may be combined using one or moredroplet operations with a lysis buffer droplet in order to yield a lysedsample droplet in which nucleic acid has been released. A droplet thatincludes magnetically responsive capture beads may be combined with thelysed sample droplet in order to bind nucleic acid, yielding a nucleicacid capture droplet in which nucleic acid is bound to the magneticallyresponsive beads. The nucleic acid capture droplet may be transportedusing one or more droplet operations into the presence of a magnet andwashed using a merge-and-split wash protocol to remove unbound material,yielding a washed bead-containing droplet substantially lacking inunbound material. In some applications, the washed bead-containingdroplet may be merged with an elution buffer droplet to elute thenucleic acid, yielding a bead-containing elution droplet. Thebead-containing elution droplet may be transported using one or moredroplet operations into a thermal zone in order to promote release ofthe nucleic acid. In other applications, the washed bead-containingdroplet may be transported using one or more droplet operations into athermal zone to promote release of the nucleic acid. The eluted nucleicacid contained in the droplet surrounding the magnetically responsivebeads may then be transported away from the magnetically responsivebeads for further processing, e.g., PCR analysis.

In an alternative embodiment, a lysis buffer droplet that includesmagnetically responsive beads may be combined using one or more dropletoperations with a sample droplet to yield a nucleic acid capture dropletin which nucleic acid is bound to the magnetically responsive beads.

In yet another embodiment, a sample droplet that includes magneticallyresponsive beads may be combined using one or more droplet operationswith a lysis buffer droplet to yield a nucleic acid capture droplet inwhich nucleic acid is bound to the magnetically responsive beads.

In yet another embodiment, a sample droplet may be combined using one ormore droplet operations with a lysis buffer droplet in order to yield alysed sample droplet. A wash buffer droplet that includes magneticallyresponsive beads may be combined with the lysed sample droplet in orderto yield a nucleic acid capture droplet in which nucleic acid is boundto the magnetically responsive beads.

In yet another embodiment, magnetically responsive beads may bepre-concentrated prior to being loaded on the droplet actuator. Forexample, as the result of off-actuator processing, analytes in (e.g.,nucleic acid) may be captured on magnetically responsive beads. Themagnetically responsive beads may, for example, be provided in thesample, a lysis solution, or a wash solution. This approach permits thebeads to be assembled into a volume which is a small part of the totalsample volume. This small volume of beads may then be loaded onto thedroplet actuator, e.g., into a reservoir for on-actuator dispensing.Dispensing may result in the production of a number of unit-sized,bead-containing droplets. The magnetic capture beads may be furtherconsolidated, as needed, on the droplet actuator for conducting adroplet-based assay protocol.

Preparation of Viral RNA

Viral RNA may be prepared using, for example, Dynabeads SILANE viral NAfrom Dynal. A droplet including Proteinase K and a viral sample may becombined using one or more droplet operations with a lysis bufferdroplet to yield a lysed sample droplet in which RNA has been released.A droplet including magnetically responsive Dynabeads may be combinedwith the lysed sample droplet to bind RNA, yielding an RNA capturedroplet in which RNA is bound to the Dynabeads. The RNA capture dropletmay be transported using one or more droplet operations into thepresence of a magnet and washed using a merge-and-split wash protocol toremove unbound material, yielding a washed bead-containing dropletsubstantially lacking in unbound material. A droplet including elutionbuffer may be merged with the washed bead-containing droplet to eluteRNA, yielding a bead-containing elution droplet. The bead-containingelution droplet may be transported using one or more droplet operationsinto a thermal zone to promote release of RNA from the Dynabeads, e.g.,by heating to approximately 70° C. The eluted RNA contained in thedroplet surrounding the Dynabeads may then be transported away from theDynabeads for further processing, e.g., for execution of a droplet basedRT-PCR protocol. Viral DNA may be prepared using, for example, DynabeadsSILANE viral NA from Dynal.

Preparation of Bacterial Genomic DNA

Bacterial genomic DNA, such as genomic DNA from Bacillus anthracis, maybe prepared using beads having an affinity for DNA. For example,Dynabeads DNA DIRECT from Dynal may be used. A droplet including lysisbuffer and magnetically responsive Dynabeads may be combined using oneor more droplet operations with a bacterial sample to yield a lysedsample droplet in which released DNA is bound to the Dynabeads. The DNAcapture droplet may be transported using one or more droplet operationsinto the presence of a magnet and washed using a merge-and-split washprotocol to remove unbound material, yielding a washed bead-containingdroplet substantially lacking in unbound material. A droplet includingresuspension buffer may be merged with the washed bead-containingdroplet, yielding a DNA/bead-containing droplet. The DNA/bead-containingdroplet is ready for further processing, e.g., for execution of adroplet based PCR protocol. Alternatively, the DNA/bead-containingdroplet may be transported using one or more droplet operations into athermal zone to promote release of DNA from the Dynabeads, e.g., byheating to approximately 65° C. The eluted DNA contained in the dropletsurrounding the Dynabeads may then be transported away from theDynabeads for further processing, e.g., for execution of a droplet basedPCR protocol.

Droplet Actuator Systems

The invention provides droplet actuators with storage and/ortransmission devices useful for controlling and/or monitoringdistribution and/or use of droplet actuators. The invention alsoprovides networked systems and methods of using such networked systemsfor controlling and/or monitoring distribution and/or use of dropletactuators.

FIG. 17 illustrates a droplet actuator device 1700 of the invention.Droplet actuator device 1700 includes droplet actuator 1724. Dropletactuator 1724 includes an electronic storage and/or transmission element1714. Electronic storage and/or transmission element 1714 may be affixedto and/or incorporated in droplet actuator 1724 or affixed to and/orincorporated in a cartridge incorporating droplet actuator 1724. Storageand/or transmission element 1714 may, for example, include semiconductormemory, magnetic storage, optical storage, and/or other available formsof computer readable data storage. Storage and/or transmission element1714 may be volatile or non-volatile. Examples of specific storageand/or transmission elements 1714 include radio-frequency identification(RFID) tags, read-only memory (ROM), random access memory (RAM),electrically erasable programmable read-only memory (EEPROM) (such asflash memory), and magnetic stripes.

In one embodiment, the storage and/or transmission element includes anRFID tag. The RFID tag may be affixed to and/or incorporated in thedroplet actuator or droplet actuator cartridge. For example, where thesubstrate of the droplet actuator is made from a printed circuit board(PCB), the RFID tag may also be mounted on the PCB. The RFID tag mayprovide for wireless identification of the droplet actuator. Forexample, the RFID tag may transmit a unique identifier for each dropletactuator. RFID monitors, such as those manufactured by Texas Instruments(Dallas, Tex.), may track the location and use of the droplet actuator.In one embodiment, the invention provides a system in which a subject'sRFID and a droplet actuator's RFID are scanned at a subject's bedsideinto a system which matches the subject with the droplet actuator. Thesubject's sample may be loaded onto the droplet actuator, for example,into a droplet actuator reservoir and/or into the droplet operations gapof the droplet actuator. The droplet actuator may be mounted on aninstrument and used to execute an assay using the sample. Uponcompletion of the assay, assay results may be automatically associatedwith the subject. In some embodiments, the information may beautomatically added to the laboratory information system or hospitalinformation system or the subject's electronic medical records. Similarmethods may be used in testing applications outside of the medicalfield.

In another example, the storage and/or transmission element includes amemory device, such as a random access memory (RAM) device, read-onlymemory (ROM) device, or a flash drive. For example, such a dropletactuator may be provided in the form of a peripheral connect device,such as a USB device, that plugs into a computer to power the dropletactuator and permit data exchange between the computer and the device.As another example, the droplet actuator can also be connected andpowered by a personal digital assistant (PDA) or a smartphone or amobile phone. Identifying information from the droplet actuator may beread by the computer, and output information from the assay may bestored on the computer and/or the USB device.

In another embodiment, the invention provides a system for conductingenvironmental studies, such as studies relating to pollution and/orbiological or chemical warfare agents. The device may include a dropletactuator in an instrument associated with the droplet actuator includingelements required to power the droplet actuator and/or control thedroplet actuator. The system may also include elements for gatheringother information, such as temperature, humidity, GPS location, and thelike. Information including the results of the assay and the otherinformation may be transmitted to a networked computer. Informationincluding the results the assay and the other information mayalternatively be stored on the droplet actuator device and informationfrom multiple devices may be transported to an uploading station wherethe information may be aggregated onto a computer or a computer network.

In one embodiment, the invention provides a system for detecting andtracking the extent of a chemical or biological attack or release of adangerous chemical. Droplet actuators may be installed at variouslocations throughout a target region, for example, on buildings, farms,water supply sources, buoys, weather balloons, etc. Droplet actuatorsmay be installed on mobile devices, such as mobile robotic devices,airplanes, unmanned drones, and vehicle fleets (such as police cars,school buses, ambulances, military vehicles, oceangoing vessels, postalvehicles, commercial vehicles, etc.). Droplet actuators may beassociated with GPS systems for determining coordinates of the dropletactuators when samples are taken. Tests may be executed using thedroplet actuator devices, and results may be transmitted back to acentral location, along with sample collection location information, foraggregation and analysis.

FIG. 18 illustrates another embodiment, droplet actuator 1724 isprovided as part of a magnetic stripe card device 1800. Card device 1800includes a card 1805 with a magnetic stripe 1810 affixed thereto forreceiving and storing data. A droplet actuator 1724 is also affixed tothe card. Droplet actuator 1724 may include electrical contacts 1815 forelectrically coupling droplet actuator 1724 to an instrument.Alternatively, droplet actuator 1724 may be electrically connected towires on card 1805. Wires on card 1805 may terminate in contacts, andthese contacts may be electrically coupled to electrical contacts on theinstrument so that the droplet actuator may be controlled by theinstrument. Droplet actuator 1724 may include an opening 1820 or loadingmechanism for loading a sample into droplet actuator 1724 in a mannerwhich subjects the sample to droplet operations mediated by electrodescoupled to the electrical contacts and controlled by the instrument towhich the card/droplet actuator is electrically coupled.

In some embodiments, the card may have the shape and size of a standardcredit card, and magnetic stripe 1810 may have a location on card 1805which is similar to the location of the magnetic stripe on a standardcredit card. Magnetic stripe 1810 may be, for example, any magneticstripe capable of storing data, such as those commonly used on magneticstripe cards (e.g., credit cards, identity cards, and transportationtickets). Magnetic stripe 1810 may be read by physical contact andswiping past a reading head (not shown), as is well known. In oneembodiment, the instrument is configured so that magnetic stripe 1810may be read as the card is inserted into the instrument. Further,information from the assay may be written to magnetic stripe 1810 duringand/or following the completion of the assay.

The instrument may be configured in a manner similar to an automatedteller machine, in which the card is inserted by a user, a card readerdevice transports the card into an operational position in which thecard electrical contacts are coupled to the instrument. Establishing thecard in operational position may be controlled by a card insertiondevice and/or may be manually controlled. Further, in operationalposition, any detection region or window on the droplet actuator may bealigned with a detector on the instrument. In operational position, theinstrument may control the execution of an assay on the dropletactuator, and then read and store information to and from the magneticstripe. Information from magnetic stripe 1810 may be read by a magneticstripe reader. An assay may be conducted, and information pertaining toassay results may be written to magnetic stripe 1810. The instrument maybe coupled to a network and may upload results from the assay to thenetwork, e.g., into an electronic medical record system. The instrumentmay include an output device, such as a display and/or printer, whichoutputs information pertaining to assay results. In another embodiment,a magnetic stripe reader/writer at a subject's bedside is used toassociate card 1800 with a specific subject, e.g., by reading a cardidentifier from magnetic stripe 1810 and copying the identifier into asubject record and/or by writing a subject identifier onto card 1800.Magnetic stripe 1810 may also include information about the expirationdate of card device 1800, information about the assay type, instructionsfor a user for electronic display by the instrument, and softwareinstructions for controlling the assay or selecting a software protocolon the instrument for controlling the assay. Further, printed materialon card device 1800 may also include information about the expirationdate of card device 1800, information about the assay type, instructionsfor a user. In an alternative embodiment, the card is a smart cardcontaining an integrated circuit actuator. The card may have metalcontacts connecting the card physically to the reader. Similarly, thecard may be a contactless card that uses a magnetic field or radiofrequency (RFID) for proximity reading. A battery supply may be includedon the card for self-contained execution of an assay.

As noted, the invention provides a droplet actuator with electronicstorage and/or transmission element. Information that may be storedand/or transmitted by the electronic storage and/or transmission elementincludes, for example, sample identification information, testidentification information (such as assay type), and subjectidentification information. Examples of subject identificationinformation include medical history information, subject contactinformation, insurance information, and test results information. Inshort, the information may include any data of interest suited for theapplication in which it is used. Systems that use the droplet actuatorsof the invention that have data storage capability may, for example,provide the advantage of automated tracking, automated distribution,reduction in medical errors, and/or improved anonymity.

FIG. 19 is a functional diagram of a sample collection and analysissystem 1900 of the invention. System 1900 utilizes droplet actuators1724 of the invention that have data storage components 1714. In thisembodiment, sample collection system 1900 includes one or more kiosks1915 for dispensing one or more droplet actuators 1724. Kiosks 1915 maybe standalone kiosks or may be provided as components of a computernetwork, such as a wide area network (WAN) or local area network (LAN).Kiosk 1915 may be located, for example, in a pharmacy, grocery store,mall, gas station, doctor's office, hospital, clinic, and/or anyconvenient location suited for collecting samples. An example method ofusing system 1900 may include, but is not limited to, the followingsteps:

Step 1: Using kiosk 1915, a subject obtains a droplet actuator 1724. Forexample, droplet actuator 1724 may be purchased by the subject using acredit card transaction. Droplet actuator 1724 is dispensed from kiosk1915. The subject may input identifying information into system 1900using kiosk 1915. Kiosk 1915 may include a keypad for inputtinginformation, information may be collected from the subject's credit cardor insurance card, and/or the subject may be provided with anidentification card with information that is readable by kiosk 1915.User information may, for example, include name, address, telephone,insurance information, physician information, etc. System 1900 mayassociate the subject's identifying information with identifyinginformation from droplet actuator 1724. A user-generated code or akiosk-generated code may be provided during the transaction. In thisway, the purchased droplet actuator 1724 may be associated with subject.This association may be stored locally within kiosk 1915 or,alternatively, the information is transferred to a networked computervia the networked system.

Step 2: The subject or a medical care provider may load a sample ondroplet actuator 1724. For example, a urine sample, blood sample, salivasample, or stool sample may be loaded into a reservoir of dropletactuator 1724. Non-medical samples may also be used, e.g., a drinkingwater sample, aquarium water sample, a swimming pool water sample, apond water sample, a plant sample, and the like. Kiosk 1915 may dispenseinstructions and or sample collection devices for collecting andhandling the sample. Droplet actuator 1724 may be sealed to preventleakage of the sample. Droplet actuator 1724 may be placed in a sealedcontainer to prevent leakage of the sample.

Step 3: The subject may return the droplet actuator 1724 that has asample therein to the site of kiosk 1915. Droplet actuator 1724 may beinserted into kiosk 1915 via any kind of receiving mechanism, such as asecure slot. Droplet actuator 1724 may be stored in a secure mannerwithin kiosk 1915 until such time that it may be removed by anattendant. Droplet actuator 1724 may be stored in a temperaturecontrolled environment within kiosk 1915. Alternatively, dropletactuator 1724 may be provided to an attendant at the site of the kiosk,at a physician's office or elsewhere. In another embodiment, a mailinglabel, package, and/or instructions may be dispensed with dropletactuator 1724, and droplet actuator may be mailed to a laboratory forprocessing.

Step 4: Droplet actuator 1724 is removed from kiosk 1915 by anattendant.

Step 5: Data storage device 1714 of droplet actuator 1724 may be scannedor otherwise read in order to extract the unique identification numberand subject information. Alternatively, data storage device 1714 mayinclude only a unique serial number, and the patient information may bestored at the local kiosk 1915 or at a centralized computerelectronically coupled to the kiosk. The subject information may thus bematched to the serial number of the droplet actuator 1724. In this way,the droplet actuator 1724 is automatically associated with the certainsubject that has provided the sample.

Step 6: Sample within droplet actuator 1724 may be analyzed and theresults automatically reported via, for example, telephone, email,and/or the subject accessing the results via kiosk 1915 (e.g., using acode). For example, results may be reported to the subject or thesubject's medical care provider.

A similar process may be used in a hospital environment. For example, asubject identifier and droplet actuator identifier may be associated ata subject's bedside or via a hospital supply system. Associatedinformation may be centrally stored and/or stored on storage and/ortransmission element 1714 of droplet actuator 1724.

A similar approach may be used for environmental studies, such astesting for contaminants in drinking water. Sample collection deviceswith unique serial numbers may be mailed to participants in the study.The serial numbers may, for example, be stored in an electronic format,such as an RFID actuator or in a physical format, such as a bar code.Users may load samples into the sample collection devices, and drop offthe samples at local collection points (or ship them to a collectionpoint) for analysis. The identifying information may be associated withthe user's address. In this manner, certain geographical distributionsof drinking water contamination may be identified. In a relatedembodiment, the users may take the sample collection devices to a kioskanalyzer, which controls droplet operations on the sample collectiondevice and provides an output directly to the user. For example, a usermay purchase a drinking water analysis collection device at a store,take it home and load it with a drinking water sample, bring it to akiosk where it can be plugged in, permit the kiosk to run tests on thedrinking water using a droplet actuator device that is part of thesample collection device, and provide the user with an output indicativeof certain drinking water contaminants.

In yet another embodiment, kiosk 1915 may be reader instrument, and theuser may insert droplet actuator 1724 into a reader slot, and kiosk 1915may execute an assay using the droplet actuator. For example, a user maybring a water sample from home, obtain a droplet actuator from kiosk1915, load a droplet of water onto the droplet actuator, electronicallycouple the droplet actuator to kiosk, whereupon kiosk 1915 executes andassay and provides the user with results. As another example, a samplecollection container may be mailed to a user, the user may collect thesample, such as a water sample, take the sample to kiosk 1915, obtain adroplet actuator from kiosk 1915, load a droplet of water onto thedroplet actuator, couple the droplet actuator to kiosk, whereupon kiosk1915 executes and assay and provides the user with results. Results mayalso be centrally stored for further analysis. In some cases, kiosk 1915may be set up to receive biohazardous materials.

FIG. 20 is a functional diagram of a sample collection system 2000 ofthe invention. System 2000 utilizes droplet actuators 1724 with datastorage components 1714 (see FIG. 17). Sample collection system 2000includes distribution center 2010, which may be, for example, amanufacturer facility or a warehouse distribution facility. Distributioncenter 2010 maintains an inventory of droplet actuators 1724.

A server is provided by which one or more subjects or medical providersmay place orders for droplet actuators 1724 via, for example, a computerin a subject's home, a medical care provider's office, pharmacy, clinic,and so on. Using any standard web browser or network interfaceapplication, the server facilitates the order placement and paymentoperations. For each transaction by which droplet actuator 1724 isordered, an association may be made between droplet actuator identifyinginformation and an ordering party's or subject's identifyinginformation. This association may be provided via credit cardinformation, purchase order number, other subject information such asname, address, email, and telephone, and/or a subject-generated orsystem-generated code. In this manner, droplet actuator 1724 may beassociated with a certain subject. This association may be stored on webserver or other networked computer and associated with assay results.

As will be appreciated by one of skill in the art, aspects of theinvention may be embodied as a method, system, or computer programproduct. Accordingly, various aspects of the invention may take the formof hardware embodiments, software embodiments (including firmware,resident software, micro-code, etc.), or embodiments combining softwareand hardware aspects that may all generally be referred to herein as a“circuit,” “module” or “system.” Furthermore, the methods of theinvention may take the form of a computer program product on acomputer-usable storage medium having computer-usable program codeembodied in the medium.

Any suitable computer useable medium may be utilized for softwareaspects of the invention. The computer-usable or computer-readablemedium may be, for example but not limited to, an electronic, magnetic,optical, electromagnetic, infrared, or semiconductor system, apparatus,device, or propagation medium. More specific examples (a non-exhaustivelist) of the computer-readable medium would include some or all of thefollowing: an electrical connection having one or more wires, a portablecomputer diskette, a hard disk, a random access memory (RAM), aread-only memory (ROM), an erasable programmable read-only memory (EPROMor Flash memory), an optical fiber, a portable compact disc read-onlymemory (CD-ROM), an optical storage device, a transmission medium suchas those supporting the Internet or an intranet, or a magnetic storagedevice. Note that the computer-usable or computer-readable medium couldeven be paper or another suitable medium upon which the program isprinted, as the program can be electronically captured, via, forinstance, optical scanning of the paper or other medium, then compiled,interpreted, or otherwise processed in a suitable manner, if necessary,and then stored in a computer memory. In the context of this document, acomputer-usable or computer-readable medium may be any medium that cancontain, store, communicate, propagate, or transport the program for useby or in connection with the instruction execution system, apparatus, ordevice.

Computer program code for carrying out operations of the invention maybe written in an object oriented programming language such as Python,Java, Smalltalk, C++ or the like. However, the computer program code forcarrying out operations of the invention may also be written inconventional procedural programming languages, such as the “C”programming language or similar programming languages. The program codemay execute entirely on the user's computer, partly on the user'scomputer, as a stand-alone software package, partly on the user'scomputer and partly on a remote computer or entirely on the remotecomputer or server. In the latter scenario, the remote computer may beconnected to the user's computer through a local area network (LAN) or awide area network (WAN), or the connection may be made to an externalcomputer (for example, through the Internet using an Internet ServiceProvider).

Certain aspects of invention are described with reference to variousmethods and method steps. It will be understood that each method stepcan be implemented by computer program instructions. These computerprogram instructions may be provided to a processor of a general purposecomputer, special purpose computer, or other programmable dataprocessing apparatus to produce a machine, such that the instructions,which execute via the processor of the computer or other programmabledata processing apparatus, create means for implementing thefunctions/acts specified in the methods.

The computer program instructions may also be stored in acomputer-readable memory that can direct a computer or otherprogrammable data processing apparatus to function in a particularmanner, such that the instructions stored in the computer-readablememory produce an article of manufacture including instruction meanswhich implement various aspects of the method steps.

The computer program instructions may also be loaded onto a computer orother programmable data processing apparatus to cause a series ofoperational steps to be performed on the computer or other programmableapparatus to produce a computer implemented process such that theinstructions which execute on the computer or other programmableapparatus provide steps for implementing various functions/actsspecified in the methods of the invention.

CONCLUDING REMARKS

The foregoing detailed description of embodiments refers to theaccompanying drawings, which illustrate specific embodiments of theinvention. Other embodiments having different structures and operationsdo not depart from the scope of the present invention. The term “theinvention” or the like is used with reference to certain specificexamples of the many alternative aspects or embodiments of theapplicants' invention set forth in this specification, and neither itsuse nor its absence is intended to limit the scope of the applicants'invention or the scope of the claims. This specification is divided intosections for the convenience of the reader only. Headings should not beconstrued as limiting of the scope of the invention. The definitions areintended as a part of the description of the invention. It will beunderstood that various details of the present invention may be changedwithout departing from the scope of the present invention. Furthermore,the foregoing description is for the purpose of illustration only, andnot for the purpose of limitation, as the present invention is definedby the claims as set forth hereinafter.

1-173. (canceled)
 174. A droplet actuator comprising: (a) two substratesseparated by a droplet operations gap; (b) one or more electrodesassociated with one or both substrates and arranged for conducting oneor more droplet operations in the droplet operations gap; (c) a reagentstorage cassette comprising one or more reservoirs comprising one ormore liquids; and (d) a fluid path from the one or more reservoirs intothe droplet operations gap, wherein the fluid path is blocked by a film.175. The droplet actuator of claim 174 further comprising one or moreplungers associated with the one or more reservoirs and arranged toforce liquid from the one or more reservoirs into the fluid path whendepressed into the one or more reservoirs.
 176. The droplet actuator ofclaim 175 comprising a series of the one or more reservoirs and a seriesof the one or more plungers, where each of the reservoirs is associatedwith a corresponding plunger arranged to force liquid from the reservoirinto the fluid path.
 177. The droplet actuator of claim 176 wherein theone or more plungers are coupled to a common plunger depressor.
 178. Thedroplet actuator of claim 175 wherein the film is selected to break uponapplication of pressure to liquid in the one or more reservoirs bydepressing the one or more plungers.
 179. The droplet actuator of claim178 wherein the film is scored to facilitate breaking of the film uponapplication of pressure to liquid in the one or more reservoirs bydepressing the one or more plungers.
 180. The droplet actuator of claim174 wherein the reagent storage cassette comprises an awl, scribe orother puncturing device arranged to puncture the film and thereby permitliquid to flow through the fluid path.
 181. The droplet actuator ofclaim 175 wherein the device comprises an awl, scribe or otherpuncturing device slidably inserted within a slot in the one or moreplungers and arranged to puncture the film and thereby permit liquid toflow through the fluid path.
 182. The droplet actuator of claim 175comprising a series of reservoirs, wherein each reservoir in the seriesof the reservoirs is associated with a connecting fluid path extendingfrom the reservoir and into a channel of the fluid path, such that upondepression of the one or more plungers, a series of droplets is forcedthrough the connecting fluid path and into the channel.
 183. The dropletactuator of claim 182 wherein the channel comprises a liquid fillerfluid which is immiscible with the series of droplets.
 184. The dropletactuator of claim 182 wherein the channel is coupled to a pressure orvacuum source for flowing droplets through the channel and into thedroplet operations gap.
 185. The droplet actuator of claim 182 whereinthe channel is associated with one or more electrodes configured fortransporting droplets through the channel and into the dropletoperations gap.
 186. The droplet actuator of claim 174 comprising aseries of reservoirs, wherein each reservoir in the series of thereservoirs is associated with a fluid path from the reservoir into thedroplet operations gap.
 187. The droplet actuator of claim 186 whereinliquid forced through the fluid path into the droplet operations gap issubject to one or more droplet operations in the droplet operations gap.188. The droplet actuator of claim 186 wherein the fluid path from thereservoir into the droplet operations gap passes through an opening inan electrode.
 189. The droplet actuator of claim 186 wherein the fluidpath is fluidly coupled with one or more filler fluid channels arrangedfor flowing filler fluid around droplets in the fluid path.
 190. Amethod comprising: (a) providing a channel; (b) flowing an immiscibleliquid including a droplet through the channel and into proximity with aset of one or more electrodes; (c) using the set of one or moreelectrodes with the droplet to conduct a droplet operation; and (d)continuing to flow the droplet or one or more daughter droplets formedduring the droplet operation through the channel.
 191. The method ofclaim 190 wherein the droplet operation is effected without stoppingflow of the immiscible liquid through the channel.
 192. The method ofclaim 190 wherein the droplet operation comprises splitting the dropletinto two or more daughter droplets.
 193. The method of claim 190 whereinthe droplet operation comprises interrupting the flow of the dropletthrough the channel.